Aerosolized fluoroquinolones and uses thereof

ABSTRACT

Disclosed herein are formulations of fluoroquinolones suitable for aerosolization and use of such formulations for aerosol administration of fluoroquinolone antimicrobials for the treatment of pulmonary bacterial infections. In particular, inhaled levofloxacin specifically formulated and delivered for bacterial infections of the lungs is described. Methods include inhalation protocols and manufacturing procedures for production and use of the compositions described.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/012,307 filed Aug. 28, 2013, which is a continuation of U.S.application Ser. No. 12/604,340 filed Oct. 22, 2009, issued as U.S. Pat.No. 8,546,423, which is a divisional of U.S. application Ser. No.11/436,875 filed May 18, 2006, issued as U.S. Pat. No. 7,838,532, whichclaims priority to U.S. Provisional Application No. 60/682,530 filed May18, 2005, U.S. Provisional Application No. 60/696,160 filed Jul. 1,2005, and U.S. Provisional Application No. 60/773,300 filed Feb. 13,2006, all of which are incorporated herein by reference in theirentireties.

BACKGROUND OF THE INVENTION

Antibiotics have been effective tools in the treatment of infectiousdiseases during the last half-century. From the development ofantimicrobial therapy to the late 1980s, most bacterial infectionsoccurring in patients in developed countries could be controlled unlessthe infection occurred in an organ or environment where antibiotics weredifficult to deliver or were ineffective, such as bacterial infectionsof the circulatory system in sepsis patients, or bacterial infections ofthe lungs in cystic fibrosis. However, even in ordinary infections, inresponse to the pressure of antimicrobial usage, multiple resistancemechanisms have become widespread and are threatening the clinicalutility of even the most aggressive antibacterial therapy. The increasein antimicrobial resistant strains has been particularly common in majorhospitals and care centers. The consequences of the increase inresistant strains include higher morbidity and mortality, longer patienthospitalization, and an increase in treatment costs.

Bacteria have developed several different mechanisms to overcome theaction of antimicrobials. These mechanisms of resistance can be specificfor a molecule or a family of antimicrobials, or can be non-specific andbe involved in resistance to unrelated antimicrobials. Severalmechanisms of resistance can exist in a single bacterial strain, andthose mechanisms may act independently or they may act synergisticallyto overcome the action of an antimicrobial or a combination ofantimicrobials. Specific mechanisms include degradation of the drug,inactivation of the drug by enzymatic.

Modification and alteration of the drug target. There are, however, moregeneral mechanisms of drug resistance, in which access of theantimicrobial to the target is prevented or reduced by decreasing thetransport of the antimicrobial into the cell or by increasing the effluxof the drug from the cell to the outside medium. Both mechanisms canlower the concentration of drug at the target site and allow bacterialsurvival in the presence of one or more antimicrobials that wouldotherwise inhibit or kill the bacterial cells. Some bacteria utilizeboth mechanisms, combining a low permeability of the cell wall(including membranes) with an active efflux of antimicrobials.

SUMMARY OF THE INVENTION

Various embodiments provide compositions and methods for optimalantimicrobial activity for the treatment of respiratory tract andpulmonary infections in human and/or veterinary subjects usingshort-term, rapid aerosol administration, and through the delivery ofhigh-concentration drug exposure directly to the affected tissue.Specifically, in some embodiments, concentrated doses of agents from thefluoroquinolone class of antibiotic are delivered to produce maximumconcentrations of active drug to the respiratory, pulmonary, and othernon-oral topical compartments including, but not limited to the skin,rectum, vagina, urethra, urinary bladder, eye, and ear. Becausedifferent drug products are known to produce different antimicrobialeffects depending on the dose, form, concentration and delivery profile,some embodiments provide specific formulation and delivery parametersthat produce antimicrobial results that are therapeutically significant.The invention includes, but is not limited to, specific fluoroquinoloneantibiotics, such as levofloxacin, formulated to enable aerosoladministration meeting specific concentrations and antimicrobialcriteria necessary to treat patients with distinct bacterial infections.These formulations and methods are useful with commercially availableinhalation devices for one or more aerosol therapeutic productopportunities.

Aerosol administration directly to the nasal, sinus, respiratory tractand pulmonary compartments through intra-nasal or oral inhalationenables high concentration drug delivery to a site of respiratoryinfection with decreased risk of extra-respiratory toxicity associatedwith non-respiratory routes of drug delivery. Furthermore, directadministration to the site of infection permits very high local druglevels, a property that enables “rapid administration, highconcentration, local exposure” killing effect special to this class ofantibiotic. Accordingly, because the microbial killing effect of aparticular antibiotic compound and therapeutic composition variesdepending on the formulation and delivery parameters, newer compositionsand delivery methods can be developed for existing drug compounds thatare re-formulated and administered through novel delivery techniques.Other topical infections may also benefit from this discovery throughhigh concentration, direct exposure of fluoroquinolone to infected skin,rectum, vagina, urethra, urinary bladder, eye, and ear.

Members of the fluoroquinolone drug class exhibit unique pharmacologicproperties, including bioavailability (F), mean absorption time (MAT)from the lung, maximal drug concentrations in the epithelial liningfluid, bronchial lavage fluid, sputum and/or lung tissue (Cmax)following aerosol administration, pulmonary retention time, area underthe curve (AUC), minimal inhibitory concentrations (MIC) of theantibiotic required for antibacterial activity, AUC/MIC ratio, and localand systemic safety. Specific to the invention is the use short-term,rapid aerosol administration, delivering high concentration drugexposure directly to the affected tissue (ELF, sputum, BAL, tissue) viaaerosol delivery for treatment of bacterial infection in animals andhumans.

In addition to the clinical and pharmacological requirements present inany composition intended for therapeutic administration, manyphysicochemical factors unique to a drug compound must also beconsidered. These include, but are not limited to aqueous solubility,viscosity, partitioning coefficient (Log P), predicted stability invarious formulations, osmolality, surface tension, pH, pKa, pKb,dissolution rate, sputum permeability, sputum binding/inactivation,taste, throat irritability and acute tolerability.

Other factors to consider when designing the product form includefluoroquinolone physical chemistry and antibacterial activity, diseaseindication, clinical acceptance, and patient compliance. By non-limitingexample, if desired the aerosol fluoroquinolone product may be in theform of a simple liquid (e.g. soluble fluoroquinolone withnon-encapsulating soluble excipients/salts), complex liquid (e.g.fluoroquinolone encapsulated or complexed with soluble excipients suchas lipids, liposomes, cyclodextrins, microencapsulations, andemulsions), complex suspension (e.g. fluoroquinolone as alow-solubility, stable nanosuspension alone, co-crystal/co-precipitatecomplexes, and mixtures with low solubility lipids such as solid-lipidnanoparticles), or dry powder (dry powder fluoroquinolone alone or inco-crystal/co-precipitate/spray-dried complex or mixture with lowsolubility excipients/salts or readily soluble blends such as lactose).

Combined with product form is a packaging consideration. By non-limitingexample, considerations for packaging include intrinsic productstability, the need for stability-providing lyophilization, deviceselection (e.g. liquid nebulizer, dry-powder inhaler, meter-doseinhaler), and packaging form (e.g. simple liquid or complex liquidformulation in a vial as liquid or lyophilisate to be dissolved prior toor upon insertion into the device; complex suspension formulations in avial as a liquid or lyophilisate with or without a solublesalt/excipient component to be dissolved prior to or upon insertion intothe device, or separate packaging of liquid and solid components; drypowder formulations in a vial, capsule or blister pack; and otherformulations packaged as readily soluble or low-solubility solid agentsin separate containers alone or together with readily soluble orlow-solubility solid agents. Any separately packaged agent will bemanufactured to be mixed prior to or upon insertion into the deliverydevice).

In some aspects, the present invention relates to the aerosol andtopical delivery of fluoroquinolone antimicrobials, such aslevofloxacin. Levofloxacin has favorable solubility characteristicsenabling dosing of clinically-desirable fluoroquinolone levels byaerosol (e.g. through liquid nebulization, dry powder dispersion ormeter-dose administration) or topically (e.g. aqueous suspension, oilypreparation or the like or as a drip, spray, suppository, salve, or anointment or the like) and can be used in methods for acute orprophylactic treatment of an infected vertebrate, e.g. a bacterialinfection, or a subject at risk of an infection.

Others include: ofloxacin, lomefloxacin, pefloxacin, ciprofloxacin,gatifloxacin, gemifloxacin, moxifloxacin, tosufloxacin, pazufloxacin,rufloxacin, fleroxacin, balofloxacin, sparfloxacin, trovafloxacin,enoxacin, norfloxacin, clinafloxacin, grepafloxacin, sitafloxacin,marbofloxacin, orbifloxacin, sarafloxacin, danofloxacin, difloxacin,enrofloxacin, garenoxacin, prulifloxacin, olamufloxacin, DX-619,TG-873870 and DW-276.

In a preferred embodiment, the method treats a bacterial infection in asubject using concentrated aerosol levofloxacin administered to asubject infected with a pathogenic bacteria in the lungs.

The therapeutic method may also include a diagnostic step, such asidentifying a patient infected with a particular pathogenic bacteria, ora resistant bacteria. In some embodiments, the method further includesidentifying a patient as colonized with a bacteria that is capable ofdeveloping resistance to fluoroquinolone antimicrobials. In someembodiments, the delivered amount of aerosol levofloxacin is sufficientto overcome resistance or prevent resistance development tolevofloxacin. In one embodiment, the MIC of the fluoroquinoloneantibacterial compound for the microbe is greater than about 2 ug/ml.

In another embodiment, the delivered amount of aerosol levofloxacin issufficient to overcome resistance or prevent further resistance of anorganism exhibiting an MIC of the fluoroquinolone antibacterial compoundthat is greater than about 4 ug/ml.

In another embodiment, the delivered amount of aerosol fluoroquinoloneis sufficient to overcome resistance or prevent further resistance of anorganism exhibiting an MIC of the fluoroquinolone antibacterial compoundthat is greater than about 8 ug/ml.

In another embodiment, the delivered amount of aerosol fluoroquinoloneis sufficient to overcome resistance or prevent further resistance of anorganism exhibiting an MIC of the fluoroquinolone antibacterial compoundthat is greater than about 16 ug/ml.

In another embodiment, the delivered amount of aerosol fluoroquinoloneis sufficient to overcome resistance or prevent further resistance of anorganism exhibiting an MIC of the fluoroquinolone antibacterial compoundthat is greater than about 32 ug/ml.

In another embodiment, a method is provided for prophylactic treatmentof a subject, including administering to a subject, susceptible tomicrobial infection or a chronic carrier of an asymptomatic or lowsymptomatic microbial infection, a fluoroquinolone antimicrobial toachieve a minimal inhibitory concentration of antimicrobial at a site ofpotential or current infection. In one embodiment, the method furthercomprising identifying a subject as a subject at risk of a bacterialinfection or at risk for an exacerbation of an infection.

In another embodiment, a method is provided for acute or prophylactictreatment of a patient through aerosol administration of fluoroquinoloneto produce and maintain threshold drug concentrations in the lung, whichmay be measured as drug levels in epithelial lining fluid (ELF), sputum,lung tissue or bronchial lavage fluid (BAL). One embodiment includes theuse of short-term, rapid aerosol administration, delivering highconcentration drug exposure directly to the affected tissue fortreatment of bacterial infections in animals and humans.

In another embodiment, a method is provided for treating a microbialinfection in a subject, including administering to a subject infectedwith a microbe a fluoroquinolone antimicrobial to achieve a minimalinhibitory concentration of antimicrobial at a site of infection. In oneembodiment, the method further comprising identifying the subject asinfected with a microbe that is resistant to an antimicrobial agent.

In another embodiment, a method is provided for acute or prophylactictreatment of a patient through non-oral or non-nasal topicaladministration of fluoroquinolone to produce and maintain threshold drugconcentrations at the site of infection or at risk of infection. Oneembodiment includes the use of short-term, rapid aerosol administration,delivering high concentration drug exposure directly to the affectedtissue for treatment or prevention of bacterial infections in skin,rectal, vaginal, urethral, ocular, and auricular tissues.

In another embodiment, a method is provided for administering afluoroquinolone antimicrobial by inhalation, wherein the inhaled liquidor dry powder aerosol has a mean particle size from about 1 micron to 10microns mass median aerodynamic diameter and a particle size geometricstandard deviation of less than or equal to about 3 microns. In anotherembodiment, the particle size is 2 microns to about 5 microns massmedian aerodynamic diameter and a particle size geometric standarddeviation of less than or equal to about 2 microns. In one embodiment,the particle size geometric standard deviation is less than or equal toabout 1.8 microns.

In some embodiments of the methods described above, fluoroquinoloneantimicrobial minimal inhibitory concentration remains at the site ofinfection for at least about a 5 minute period, at least about a 10 minperiod, at least about a 20 min period, at least about a 30 min period,at least about a 1 hour period, 2 hour period, at least about a 4 hourperiod or other time values on the quarter hour interval. The effectivefluoroquinolone antimicrobial minimal inhibitory concentration (MIC) issufficient to cause a therapeutic effect and the effect may be localizedto the site of infection. In some embodiments, one or more levofloxacinadministrations achieve an ELF, BAL, and/or sputum fluoroquinoloneconcentration of at least 1-fold to 5000-fold the infecting orpotentially infecting organisms MIC, including all integral valuestherein such as 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold,128-fold, 256-fold, 512-fold, 1028-fold, 2056-fold, and 4112-fold themicrobials MIC.

In some embodiments, such as a pulmonary site, the fluoroquinoloneantimicrobial is administered in one or more administrations so as toachieve a respirable delivered dose daily of at least about 5 mg toabout 50 mg, including all integral values therein such as 10, 15, 20,25, 30, 35, 40 and 45 milligrams. Similarly, the fluoroquinoloneantimicrobial is administered in one or more administrations so as toachieve a respirable delivered dose daily of at least about 50 to about100 mg including all integral values therein, such as 55, 60, 65, 70,75, 80, 85, 90, and 95 mg. In some embodiments of the methods describedabove, the fluoroquinolone antimicrobial is administered in one or moreadministrations so as to achieve a respirable delivered daily dose of upto 150 mg including all integral values therein such as 105, 110, 115,120, 125, 130, 135, 140 and 145 mg. The fluoroquinolone antimicrobial isadministered in the described respirable delivered dose in less than 20minutes, less than 10 minutes, less than 7 minutes, less than 5 minutes,in less than 3 minutes and in less than 2 minutes. In some embodimentsof the methods described above, the antimicrobial agent is selected fromthe group consisting of ofloxacin, lomefloxacin, pefloxacin,ciprofloxacin, gatifloxacin, gemifloxacin, moxifloxacin, tosufloxacin,pazufloxacin, rufloxacin, fleroxacin, balofloxacin, sparfloxacin,trovafloxacin, enoxacin, norfloxacin, clinafloxacin, grepafloxacin,sitafloxacin, marbofloxacin, orbifloxacin, sarafloxacin, danofloxacin,difloxacin, enrofloxacin, garenoxacin, prulifloxacin, olamufloxacin,DX-619, TG-873870 and DW-276, although levofloxacin is preferred.

In some embodiments of the methods described above, the bacteria is agram-negative bacteria such as Pseudomonas aeruginosa, Pseudomonasfluorescens, Pseudomonas acidovorans, Pseudomonas alcaligenes,Pseudomonas putida, Stenotrophomonas maltophilia, Burkholderia cepacia,Aeromonas hydrophilia, Escherichia coli, Citrobacter freundii,Salmonella typhimurium, Salmonella typhi, Salmonella paratyphi,Salmonella enteritidis, Shigella dysenteriae, Shigella flexneri,Shigella sonnei, Enterobacter cloacae, Enterobacter aerogenes,Klebsiella pneumoniae, Klebsiella oxytoca, Serratia marcescens,Francisella tularensis, Morganella morganii, Proteus mirabilis, Proteusvulgaris, Providencia alcalifaciens, Providencia rettgeri, Providenciastuartii, Acinetobacter calcoaceticus, Acinetobacter haemolyticus,Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis,Yersinia intermedia, Bordetella pertussis, Bordetella parapertussis,Bordetella bronchiseptica, Haemophilus influenzae, Haemophilusparainfluenzae, Haemophilus haemolyticus, Haemophilus parahaemolyticus,Haemophilus ducreyi, Pasteurella multocida, Pasteurella haemolytica,Branhamella catarrhalis, Helicobacter pylori, Campylobacter fetus,Campylobacter jejuni, Campylobacter coli, Borrelia burgdorferi, Vibriocholerae, Vibrio parahaemolyticus, Legionella pneumophila, Listeriamonocytogenes, Neisseria gonorrhoeae, Neisseria meningitidis, Kingella,Moraxella, Gardnerella vaginalis, Bacteroides fragilis, Bacteroidesdistasonis, Bacteroides 3452A homology group, Bacteroides vulgatus,Bacteroides ovalus, Bacteroides thetaiotaomicron, Bacteroides uniformis,Bacteroides eggerthii, and Bacteroides splanchnicus. In some embodimentsof the methods described above, the bacteria is a gram-negativeanaerobic bacteria, by non-limiting example these include Bacteroidesfragilis, Bacteroides distasonis, Bacteroides 3452A homology group,Bacteroides vulgatus, Bacteroides ovalus, Bacteroides thetaiotaomicron,Bacteroides uniformis, Bacteroides eggerthii, and Bacteroidessplanchnicus. In some embodiments of the methods described above, thebacteria is a gram-positive bacteria, by non-limiting example theseinclude: Corynebacterium diphtherias, Corynebacterium ulcerans,Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcuspyogenes, Streptococcus milleri; Streptococcus (Group G); Streptococcus(Group C/F); Enterococcus faecalis, Enterococcus faecium, Staphylococcusaureus, Staphylococcus epidermidis, Staphylococcus saprophyticus,Staphylococcus intermedius, Staphylococcus hyicus subsp. hyicus,Staphylococcus haemolyticus, Staphylococcus hominis, and Staphylococcussaccharolyticus. In some embodiments of the methods described above, thebacteria is a gram-positive anaerobic bacteria, by non-limiting examplethese include Clostridium difficile, Clostridium perfringens,Clostridium tetini, and Clostridium botulinum. In some embodiments ofthe methods described above, the bacteria is a acid-fast bacteria, bynon-limiting example these include Mycobacterium tuberculosis,Mycobacterium avium, Mycobacterium intracellulare, and Mycobacteriumleprae. In some embodiments of the methods described above, the bacteriais a atypical bacteria, by non-limiting example these include Chlamydiapneumoniae and Mycoplasma pneumoniae.

In some embodiments of the methods described above, the subject is ahuman. In some embodiments of the methods described above, the subjectis a human with cystic fibrosis. In some embodiments of the methodsdescribed above, the subject is a human with pneumonia, a chronicobstructive pulmonary disease, or sinusitis, or a human beingmechanically ventilatated.

In another embodiment, a pharmaceutical composition is provided thatincludes a simple liquid fluoroquinolone antimicrobial formulation (e.g.soluble fluoroquinolone with non-encapsulating water soluble excipients)as described above having an osmolality from about 200 mOsmol/kg toabout 1250 mOsmol/kg. In one such embodiment, the solution has apermeant ion concentration from about 30 mM to about 300 mM. In oneembodiment, the osmolality is from about 250 mOsmol/kg to about 1050mOsmol/kg. In one embodiment, the osmolality is from prefereably fromabout 350 mOsmol/kg and about 750 mOsmol/kg and most preferablyapproximately 300 mOsmol/kg.

In another embodiment, a pharmaceutical composition is provided thatincludes a simple liquid fluoroquinolone antimicrobial formulationhaving a permeant ion concentration between from about 30 mM to about300 mM and preferably between from about 50 mM to 200 mM. In one suchembodiment, one or more permeant ions in the composition are selectedfrom the group consisting of chloride and bromide.

In another embodiment, a pharmaceutical composition is provided thatincludes a complex liquid fluoroquinolone antimicrobial formulation(e.g. fluoroquinolone encapsulated or complexed with water solubleexcipients such as lipids, liposomes, cyclodextrins,microencapsulations, and emulsions) as described above having a solutionosmolality from about 200 mOsmol/kg to about 1250 mOsmol/kg. In one suchembodiment, the solution has a permeant ion concentration from about 30mM to about 300 mM. In one embodiment, the osmolality is from about 250mOsmol/kg to about 1050 mOsmol/kg. In one embodiment, the osmolality isfrom preferably from about 350 mOsmol/kg and about 750 mOsmol/kg andmost preferably approximately 300 mOsmol/kg.

In another embodiment, a pharmaceutical composition is provided thatincludes a complex liquid fluoroquinolone antimicrobial formulationhaving a permeant ion concentration from about 30 mM to about 300 mM. Inone such embodiment, one or more permeant ions in the composition areselected from the group consisting of chloride and bromide.

In another embodiment, a pharmaceutical composition is provided thatincludes a complex liquid fluoroquinolone antimicrobial formulationhaving a permeant ion concentration from about 50 mM to about 200 mM. Inone such embodiment, one or more permeant ions in the composition areselected from the group consisting of chloride and bromide.

In another embodiment, a pharmaceutical composition is provided thatincludes a complex liquid fluoroquinolone antimicrobial formulation(e.g. fluoroquinolone as a low water soluble stable nanosuspension aloneor in co-crystal/co-precipitate complexes, or mixtures with lowsolubility lipids, such as lipid nanosuspensions) as described abovehaving a solution osmolality from about 200 mOsmol/kg to about 1250mOsmol/kg. In one such embodiment, the solution has a permeant ionconcentration from about 30 mM to about 300 mM. In one embodiment, theosmolality is from about 250 mOsmol/kg to about 1050 mOsmol/kg. In oneembodiment, the osmolality is from prefereably from about 350 mOsmol/kgand about 750 mOsmol/kg and most preferably approximately 300 mOsmol/kg.

In another embodiment, a pharmaceutical composition is provided thatincludes a complex suspension fluoroquinolone antimicrobial formulationhaving a permeant ion concentration from about 30 mM to about 300 mM. Inone such embodiment, one or more permeant ions in the composition areselected from the group consisting of chloride and bromide.

In another embodiment, a pharmaceutical composition is provided thatincludes a complex suspension fluoroquinolone antimicrobial formulationhaving a permeant ion concentration from about 50 mM to about 200 mM. Inone such embodiment, one or more permeant ions in the composition areselected from the group consisting of chloride and bromide.

In another embodiment, a pharmaceutical composition is provided thatincludes a taste-masking agent. By non-limiting example a taste-maskingagent may include a sugar, a divalent or trivalent cation that complexeswith a fluoroquinolone, optimized osmolality, and/or an optimizedpermeant ion concentration.

In another embodiment, a pharmaceutical composition is provided thatincludes a simple dry powder fluoroquinolone antimicrobial compound(e.g. fluoroquinolone alone in dry powder form with or without ablending agent such as lactose).

In another embodiment, a pharmaceutical composition is provided thatincludes a complex dry powder fluoroquinolone antimicrobial formulation(e.g. fluoroquinolone in co-crystal/co-precipitate/spray dried complexor mixture with low water soluble excipients/salts in dry powder formwith or without a blending agent such as lactose).

In another embodiment, a system is provided for administering afluoroquinolone antimicrobial that includes a container comprising asolution of a fluoroquinolone antimicrobial and a nebulizer physicallycoupled or co-packaged with the container and adapted to produce anaerosol of the solution having a particle size from about 2 microns toabout 5 microns mean mass aerodynamic diameter and a particle sizegeometric standard deviation of less than or equal to about 2.5 micronsmean mass aerodynamic diameter. In one embodiment, the particle sizegeometric standard deviation is less than or equal to about 2.0 microns.In one embodiment, the particle size geometric standard deviation isless than or equal to about 1.8 microns.

In another embodiment, a system is provided for administering afluoroquinolone antimicrobial that includes a container comprising a drypowder of a fluoroquinolone antimicrobial and a dry powder inhalercoupled to the container and adapted to produce a dispersed dry powderaerosol having a particle size from about 2 microns to about 5 micronsmean mass aerodynamic and a particle size standard deviation of lessthan or equal to about 3.0 microns. In one embodiment, the particle sizestandard deviation is less than or equal to about 2.5 microns. In oneembodiment, the particle size standard deviation is less than or equalto about 2.0 microns.

In another embodiment, a kit is provided that includes a containercomprising a pharmaceutical formulation comprising a quinoloneantimicrobial agent and an aerosolizer adapted to aerosolize thepharmaceutical formulation and deliver it to the lower respiratory tractand pulmonary compartment following intraoral administration. Theformulation may also be delivered as a dry powder or through ametered-dose inhaler.

In another embodiment, a kit is provided that includes a containercomprising a pharmaceutical formulation comprising a quinoloneantimicrobial agent and an aerosolizer adapted to aerosolize thepharmaceutical formulation and deliver it to nasal cavity followingintranasal administration. The formulation may also be delivered as adry powder or through a metered-dose inhaler.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed.

DESCRIPTION OF FIGURES

FIG. 1 is a graph showing the dose: MIC relationship of fluoroquinolonesand other antibiotics to bacterial killing.

FIG. 2 is a graph showing ciprofloxacin serum concentrations followingoral dosing in both CF patients vs healthy controls.

FIG. 3 is a graph showing ciprofloxacin sputum and serum concentrationsfollowing oral dosing.

FIG. 4A is a graph showing Levofloxacin time-kill affects on logarithmicPAM1020 cells.

FIG. 4B is a graph showing Levofloxacin time-kill affects on logarithmicPAM1032 cells.

FIG. 5A is a graph showing Levofloxacin time-kill affects on stationaryphase PAM1020 cells.

FIG. 5B is a graph showing Levofloxacin time-kill affects on stationaryphase PAM1032 cells.

FIG. 6A is a graph showing PAM 1020 re-growth following a 10 minuteLevofloxacin exposure.

FIG. 6B is a graph showing PAM 1020 re-growth following a 160 minuteLevofloxacin exposure.

FIG. 6C is a graph showing PAM 1032 re-growth following a 10 minuteLevofloxacin exposure.

FIG. 6D is a graph showing PAM 1032 re-growth following a 160 minuteLevofloxacin exposure.

FIG. 7A is a graph showing Levofloxacin time-kill affects onlate-logarithmic PAM 1020 cells under oxygen limiting conditions.

FIG. 7B is a graph showing Levofloxacin time-kill affects onlate-logarithmic PAM 1032 cells under oxygen limiting conditions.

FIG. 8A is a graph showing Levofloxacin killing kinetics of PAM1032 inMeuller-Hinton broth (MHB).

FIG. 8B is a graph showing Levofloxacin killing kinetics of PAM1032 incystic fibrosis sputum.

FIG. 9 is a graph showing levofloxacin killing affects on Pseudomonasbiofilms.

FIG. 10 is a graph showing the bactericidal effects of levofloxacin witha Cmax of 1000 μg/ml and a 10 minute half-life in a hollow fiber model.

FIG. 11 is a graph showing the bactericidal effects of levofloxacin witha Cmax of 600 μg/ml and a 10 minute half-life in a hollow fiber model.

FIG. 12 is a graph relating the micronization pressure used tomicronized dry powder levofloxacin vs. mean Levofloxacin dry powderparticle size.

FIGS. 13A and 13B are graphs showing the DSC profile of micronized andpre-micronized dry powder Levofloxacin, respectively.

FIG. 14A is a graph showing SEM photomicrographs of pre-micronized drypowder Levofloxacin.

FIG. 14B is a graph showing SEM photomicrographs of micronized drypowder Levofloxacin.

FIGS. 15A and 15B are graphs showing X-ray diffraction of pre-micronizedand micronized dry powder Levofloxacin, respectively.

FIG. 16 is a graph showing the pH solubility profile of Levofloxacin byacid titration.

FIG. 17 is a graph measuring pH while titrating Levofloxacin with HCl.

FIG. 18 is a graph showing the Vt[OH] vs. Vt of Levofloxacin.

FIG. 19 is a graph measuring pH while titrating Levofloxacin with NaOH.

FIG. 20 is a graph measuring dpH/dV vs volume of NaOH titrant (Vt) fortitration of Levofloxacin.

FIG. 21 is a graph measuring the absorbance of a Levofloxacin solutionat 257 nm vs pH.

FIGS. 22A, 22B, 22C, and 22D depict graphs showing DSC scans of pamoicacid, Levofloxacin, Levofloxacin pamoic acid co-crystallizedprecipitate, and Levofloxacin-pamoic acid physical mixture,respectively.

FIGS. 23A, 23B, 23C, and 23D depict graphs showing FTIR spectra ofpamoic acid, Levofloxacin, Levofloxacin Pamoic Acid Co-CrystallizedPrecipitate, and Levofloxacin-Pamoic Acid Physical Mixture,respectively.

FIGS. 24A and 24B depict graphs showing DSC scans of xinafoic acid, andLevofloxacin xinafoic acid co-crystallized precipitate, respectively.

FIGS. 25A and 25B depict graphs showing FTIR spectra of xinafoic acidand Levofloxacin xinafoate co-crystals, respectively.

FIGS. 26A, 26B and 26C depict graphs showing DSC scans of stearic acid,Levofloxacin stearic acid co-crystallized precipitate, and a physicalmixture of Levofloxacin and stearic acid, respectively.

FIGS. 27A, 27B, and 27C depict graphs showing FTIR spectra of stearicacid, Levofloxacin stearic acid co-crystallized precipitate, and aphysical mixture of Levofloxacin and stearic acid, respectively.

FIGS. 28A, 28B, 28C, 28D, and 28E depict graphs showing DSC scans ofoleic acid, Levofloxacin oleic acid co-crystallized precipitate,physical mixture of Levofloxacin and oleic acid (50:50), physicalmixture of Levofloxacin and oleic acid (10:90), and physical mixture ofLevofloxacin and oleic acid (90:10), respectively.

FIGS. 29A, 29B, and 29C depict graphs showing FTIR spectra of oleicAcid, Levofloxacin oleic acid co-crystallized precipitate, Levofloxacinoleic acid co-crystallized precipitate as compared to an equimolarphysical mixture of levofloxacin and oleic acid, respectively.

FIG. 30 is a graph showing the kinetic solubility of the co-crystallizedprecipitate of Levofloxacin with oleic acid at room temperature, 40° C.,and equimolar physical mixture at 40° C.

FIG. 31 is a graph showing the dissolution profile of Levofloxacinxinafoate.

FIG. 32 is a graph showing the dissolution profile of Levofloxacinxinafoate focused on the period between two and ten minutes.

FIG. 33 is a graph showing the dissolution profile of Levofloxacinxinafoate focused on the period between ten and thirty minutes.

FIG. 34 is a graph showing the dissolution profile of Levofloxacin base.

FIG. 35 is a graph showing the dissolution profile of Levofloxacinpamoate.

FIG. 36 is a graph showing the dissolution profile of Levofloxacinpamoate focused on the period between two and ten minutes.

FIG. 37 is a graph showing the dissolution profile of Levofloxacinpamoate focused on the period between ten and sixty minutes.

FIG. 38 is a graph showing the dissolution profile of Levofloxacinstearate.

FIG. 39 is a graph showing the dissolution profile of Levofloxacinstearate focused on the period between two and ten minutes.

FIG. 40 is a graph showing the dissolution profile of Levofloxacinstearate focused on the period between ten and thirty minutes.

FIG. 41 is a graph showing the complexation of Levofloxacin withdivalent and trivalent cations.

FIG. 42 is a graph showing the dual titration complexation ofLevofloxacin with Mg2+.

FIG. 43 is a graph showing the dual titration complexation ofLevofloxacin with Fe2+.

FIG. 44 is a graph showing the dual titration complexation ofLevofloxacin with Ca2+.

FIG. 45 is a graph showing the dual titration complexation ofLevofloxacin with Zn2+.

FIGS. 46A and 46B are graphs showing Levofloxacin complexed with Ca2+vs. free Levofloxacin where n=1, and where n=2, respectively.

FIGS. 47A and 47B are graphs showing Levofloxacin complexed with Mg2+vs. free Levofloxacin where n=1, and where n=2, respectively.

FIGS. 48A and 48B are graphs showing Levofloxacin complexed with Fe2+vs. free Levofloxacin where n=1, and where n=2, respectively.

FIGS. 49A and 49B are graphs showing Levofloxacin complexed with Zn2+vs. free Levofloxacin where n=1, and where n=2, respectively.

FIG. 50 is a graph showing the solubility of Levofloxacin in thepresence of Mg2+.

FIG. 51 is a graph showing the solubility of Levofloxacin in thepresence of Mg2+ at constant ionic strength.

FIG. 52 is a graph showing complexation of Levofloxacin with Fe2+ asmeasured by spectrofluorometry.

FIG. 53 is a graph showing complexation of Levofloxacin with Zn2+ asmeasured by spectrofluorometry.

DETAILED DESCRIPTION

Many of the problems associated with antimicrobial-resistant pathogenscould be alleviated if the concentration of the antimicrobial could besafely increased at the site of infection. For example, pulmonaryinfections may be treated by administration of the antimicrobial agentdirectly, at high concentrations directly to the site of infectionwithout incurring large systemic concentrations of the antimcirobial.Accordingly, some embodiments disclosed herein are improved methods fordelivering drug compositions to treat pulmonary bacterial infections.More specifically, as described herein, it has been discovered thataerosol levofloxacin and other fluoroquinolones can be safely deliveredby inhalation at levels sufficient to kill susceptible bacterialinfections, to decrease the frequencey of antimicrobial resistance andto increase efficacy against resistant pulmonary infections.

DEFINITIONS

The term “administration” or “administering” refers to a method ofgiving a dosage of an antimicrobial pharmaceutical composition to avertebrate. The preferred method of administration can vary depending onvarious factors, e.g., the components of the pharmaceutical composition,the site of the potential or actual bacterial infection, the microbeinvolved, and the severity of an actual microbial infection.

A “carrier” or “excipient” is a compound or material used to facilitateadministration of the compound, for example, to increase the solubilityof the compound. Solid carriers include, e.g., starch, lactose,dicalcium phosphate, sucrose, and kaolin. Liquid carriers include, e.g.,sterile water, saline, buffers, non-ionic surfactants, and edible oilssuch as oil, peanut and sesame oils. In addition, various adjuvants suchas are commonly used in the art may be included. These and other suchcompounds are described in the literature, e.g., in the Merck Index,Merck & Company, Rahway, N.J. Considerations for the inclusion ofvarious components in pharmaceutical compositions are described, e.g.,in Gilman et al. (Eds.) (1990); Goodman and Gilman's: ThePharmacological Basis of Therapeutics, 8th Ed., Pergamon Press.

A “diagnostic” as used herein is a compound, method, system, or devicethat assists in the identification and characterization of a health ordisease state. The diagnostic can be used in standard assays as is knownin the art.

The term “mammal” is used in its usual biological sense. Thus, itspecifically includes humans, cattle, horses, dogs, and cats, but alsoincludes many other species.

The term “microbial infection” refers to the undesired proliferation orpresence of invasion of pathogenic microbes in a host organism. Thisincludes the excessive growth of microbes that are normally present inor on the body of a mammal or other organism. More generally, amicrobial infection can be any situation in which the presence of amicrobial population(s) is damaging to a host mammal. Thus, a microbialinfection exists when excessive numbers of a microbial population arepresent in or on a mammal's body, or when the effects of the presence ofa microbial population(s) is damaging the cells or other tissue of amammal.

The term “pharmaceutically acceptable carrier” or “pharmaceuticallyacceptable excipient” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents and the like. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions.

The term “pharmaceutically acceptable salt” refers to salts that retainthe biological effectiveness and properties of the compounds of thisinvention and, which are not biologically or otherwise undesirable. Inmany cases, the compounds of this invention are capable of forming acidand/or base salts by virtue of the presence of amino and/or carboxylgroups or groups similar thereto. Pharmaceutically acceptable acidaddition salts can be formed with inorganic acids and organic acids.Inorganic acids from which salts can be derived include, for example,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. Organic acids from which salts can bederived include, for example, acetic acid, propionic acid, naphtoicacid, oleic acid, palmitic acid, pamoic (emboic) acid, stearic acid,glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid,succinic acid, fumaric acid, tartaric acid, citric acid, ascorbic acid,glucoheptonic acid, glucuronic acid, lactic acid, lactobioic acid,tartaric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicylic acid, and the like. Pharmaceutically acceptable base additionsalts can be formed with inorganic and organic bases. Inorganic basesfrom which salts can be derived include, for example, sodium, potassium,lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese,aluminum, and the like; particularly preferred are the ammonium,potassium, sodium, calcium and magnesium salts. Organic bases from whichsalts can be derived include, for example, primary, secondary, andtertiary amines, substituted amines including naturally occurringsubstituted amines, cyclic amines, basic ion exchange resins, and thelike, specifically such as isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, histidine, arginine, lysine, benethamine,N-methyl-glucamine, and ethanolamine. Other acids include dodecylsufuricacid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, andsaccharin.

“Solvate” refers to the compound formed by the interaction of a solventand fluoroquinolone antimicrobial, a metabolite, or salt thereof.Suitable solvates are pharmaceutically acceptable solvates includinghydrates.

In the context of the response of a microbe, such as a bacterium, to anantimicrobial agent, the term “susceptibility” refers to the sensitivityof the microbe for the presence of the antimicrobial agent. So, toincrease the susceptibility means that the microbe will be inhibited bya lower concentration of the antimicrobial agent in the mediumsurrounding the microbial cells. This is equivalent to saying that themicrobe is more sensitive to the antimicrobial agent. In most cases theminimum inhibitory concentration (MIC) of that antimicrobial agent willhave been reduced.

By “therapeutically effective amount” or “pharmaceutically effectiveamount” is meant a fluoroquinolone antimicrobial agent, as disclosed forthis invention, which has a therapeutic effect. The doses offluoroquinolone antimicrobial agent which are useful in treatment aretherapeutically effective amounts. Thus, as used herein, atherapeutically effective amount means those amounts of fluoroquinoloneantimicrobial agent which produce the desired therapeutic effect asjudged by clinical trial results and/or model animal infection studies.In particular embodiments, the fluoroquinolone antimicrobial agent areadministered in a predetermined dose, and thus a therapeuticallyeffective amount would be an amount of the dose administered. Thisamount and the amount of the fluoroquinolone antimicrobial agent can beroutinely determined by one of skill in the art, and will vary,depending on several factors, such as the particular microbial straininvolved. This amount can further depend upon the patient's height,weight, sex, age and medical history. For prophylactic treatments, atherapeutically effective amount is that amount which would be effectiveto prevent a microbial infection.

A “therapeutic effect” relieves, to some extent, one or more of thesymptoms of the infection, and includes curing an infection. “Curing”means that the symptoms of active infection are eliminated, includingthe total or substantial elimination of excessive members of viablemicrobe of those involved in the infection to a point at or below thethreshold of detection by traditional measurements. However, certainlong-term or permanent effects of the infection may exist even after acure is obtained (such as extensive tissue damage). As used herein, a“therapeutic effect” is defined as a statistically significant reductionin bacterial load in a host, emergence of resistance, or improvement ininfection symptoms as measured by human clinical results or animalstudies.

“Treat,” “treatment,” or “treating,” as used herein refers toadministering a pharmaceutical composition for prophylactic and/ortherapeutic purposes. The term “prophylactic treatment” refers totreating a patient who is not yet infected, but who is susceptible to,or otherwise at risk of, a particular infection. The term “therapeutictreatment” refers to administering treatment to a patient alreadysuffering from an infection. Thus, in preferred embodiments, treating isthe administration to a mammal (either for therapeutic or prophylacticpurposes) of therapeutically effective amounts of a fluoroquinoloneantimicrobial agent.

Pharmacokinetics (PK) is concerned with the time course of antimicrobialconcentration in the body. Pharmacodynamics (PD) is concerned with therelationship between pharmacokinetics and the antimicrobial efficacy invivo. PK/PD parameters correlate antimicrobial exposure withantimicrobial activity. The rate of killing by antimicrobial isdependent on antimicrobial mode of action and is determined by eitherthe length of time necessary to kill (time-dependent) or the effect ofincreasing concentrations (concentration-dependent). Accordingly, topredict the therapeutic efficacy of antimicrobials with diversemechanisms of action different PK/PD parameters may be used.

“AUC/MIC ratio” is one example of a PK/PD parameter. AUC is defined asthe area under the plasma or site-of-infection concentration-time curveof an antimicrobial agent in vivo (in animal or human). AUC/MIC ratio isdetermined by dividing the 24-hour-AUC for an individual antimicrobialby the MIC for the same antimicrobial determined in vitro. Activity ofantimicrobials with the dose-dependent killing (such asfluoroquinolones) is well predicted by the magnitude of the AUC/MICratio.

“Cmax:MIC” ratio is another PK:PD parameter. It describes the maximumdrug concentration in plasma or tissue relative to the MIC.Fluoroquinolones and aminoglycosides are examples where Cmax:MIC maypredict in vivo bacterial killing where resitance can be suppressed.

“Time above MIC” (T>MIC) is another PK/PD parameter. It is expressed apercentage of a dosage interval in which the plasma or site-of-infectionlevel exceeds the MIC. Activity of antimicrobials with thetime-dependent killing (such as beta-lactams or oxazolidinones) is wellpredicted by the magnitude of the T>MIC ratio.

The term “dosing interval” refers to the time between administrations ofthe two sequential doses of a pharmaceutical's during multiple dosingregimens. For example, in the case of ciprofloxacin, which isadministered twice daily (traditional regimen of 400 mg b.i.d) andlevofloxacin, which is administered once a day (500 mg or 750 mg q.d.),the dosing intervals are 12 hours and 24 hours, respectively.

As used herein, the “peak period” of a pharmaceutical's in vivoconcentration is defined as that time of the pharmaceutical dosinginterval when the pharmaceutical concentration is not less than 50% ofits maximum plasma or site-of-infection concentration. In someembodiments, “peak period” is used to describe an interval ofantimicrobial dosing.

The “respirable delivered dose” is the amount of drug inhaled during theinspiratory phase of the breath simulator that is equal to or less than5 microns using a simulator programmed to the European Standard patternof 15 breaths per minute, with an inspiration to expiration ratio of1:1.

Advantages of Inhaled Aerosol and Topical (Non-Oral) FluoroquinoloneDelivery

The antibiotic rate of killing is dependent upon antibiotic mode ofaction and is determined by either the length of time necessary for theantibiotic to kill (time-dependent) or the effect of increasing theantibiotic concentration (concentration-dependent). Fluoroquinolones arecharacterized by concentration-dependent, time-kill activity where atherapeutic effect requires a high local peak concentration above theMICs of the infecting pathogen.

Fluoroquinolone efficacy in humans, animals and in vitro models ofinfection is linked to AUC:MIC ratio and Cmax:MIC ratio. Given the prioruncertainty of the pharmacokinetics of fluoroquinolones in pulmonarytissue, a number of in vitro studies have been conducted to determine ifhigh doses of levofloxacin with extremely short half-lives (as predictedfrom a rat and human PK model) result in bacterial killing superior tothat seen under conditions with more prolonged residence times. In thesestudies, levofloxacin concentrations that were 0.018-fold-1024-fold theMIC were evaluated in a standard kill-curve and in vitro hollow fiberassay. In both of these assays, high concentrations of levofloxacin wererapidly bactericidal and reached their maximum level of killing in 10-20minutes. This level of killing was sustained whether levofloxacin wasmaintained at that level or given a half-life of 10 minutes.Accordingly, high doses and rapid delivery of specially formulatedlevofloxacin, such as a rapidly delivered 20-50 mg respirable depositedaerosol levofloxacin dose (which will produce initial ELF concentrationsof 800-1600 ug/mL) is rapidly bactericidal for susceptible organisms andresistant organisms with MICs up to 32 ug/ml. It is expected that theseunique antimicrobial properties of fluoroquinolones will also translateto topical administrations, including, but not limited to infections orprophylaxis of the skin, eye, ear, rectum, vagina, or urinary tract.

To measure the efficacy of different delivery models, AUCshape-enhancing levofloxacin formulations were prepared and measured invivo in comparison to non-AUC shape-enhancing levofloxacin formulationsand other antibiotics using both rat PK and mouse efficacy followingintratracheal administration. As was previously shown in a rat system,there were differences among drugs in pulmonary pharmacokinetics, withsome agents showing lower AUCs (e.g., levofloxacin), while others suchas gemifloxacin or tobramycin show higher concentrations resulting fromslower pulmonary clearance. Studies in a single dose mouse model ofinfection with aerosol doses have shown variable efficacy among thecompounds. Referring to FIG. 1, analysis of the data by dividing theaerosolized dose by the MIC indicated a strong correlation betweenDose:MIC ratio and bactericidal activity (R2=0.89). These data suggestthat the initial bactericidal activity in this model is not affected bythe pulmonary clearance of the drug. Although pulmonary clearances havenot been estimated in mice, transformation of dose to AUC using scaledrat values would be expected to degrade the relationship. Therefore,this data suggests that optimizing the AUC shape for levofloxacin maynot be necessary for aerosol levofloxacin to be effective in treatingrespiratory tract and pulmonary infections.

Recent investigations with fluoroquinolones resulted in development ofthe concept of a “mutant selection window” (MSW) for bacterialresistance arising during therapy. This concept assists in identifying aconcentration range where mutants are selected more frequently in vitroand in vivo. The lower boundary of the window is the lowestconcentration that kills the majority of infecting cells (approximatedby the MIC), while the upper boundary of the window is the drugconcentration that blocks the growth of the least susceptible first-stepmutant. Above the upper boundary concentration the growth of theinfecting bacteria requires the presence of at least two resistancemutations. This upper boundary is designated the mutant preventionconcentration (MPC). The values of MPC vary depending on bacteria andfluoroquinolone, and may be 10- to 20-fold higher than the MIC. Severalmodeling studies have demonstrated that the longer the drugconcentration exceeds the MPC at the site of infection, the moreeffectively the treatment will prevent resistance development.Conversely, the longer the antibiotic concentration stays within theMSW, the higher the probability to select resistant mutants.Importantly, the currently approved dosing regimen for oral orintravenous levofloxacin has placed this antibiotic within the MSW formore than 20% of the dosing interval for such pathogens as P. aeruginosa(Pa) and S. pneumonia. Accordingly, a high level of levofloxacinresistance is reported for both of these pathogens.

Therefore, in one embodiment, the concentration of levofloxacin at thesite of infection is increased by delivering it directly to the lungusing inhalation therapy, thereby decreasing the amount of timelevofloxacin is in the MSW. Such a therapeutic approach achieves broadercoverage of pathogens (including levofloxacin resistant strains),prevents further resistance development, and results in shorter coursesof levofloxacin therapy.

Pharmacokinetics of Orally Adminsitered Fluoroquinolones in Non-CF andCF Populations

Sputum Concentrations in CF Patients

The pharmacokinetics of ciprofloxacin has been extensively studied in CFpatients after oral administration. In fact, it has been demonstratedthat the serum PK profile of ciprofloxacin is very similar in CFpatients to healthy volunteers (FIG. 2).

Moreover, the sputum vs time profile of ciprofloxacin is very similar toits serum profile after oral administration (FIG. 3). After a 750 mgoral dose, peak concentrations of ˜4.2 μg/ml and ˜3.5 μg/ml wereachieved for serum and sputum, respectively. Serum and sputum drugconcentrations peaked at 1.5 and 4 hours, respectively. While the totalamount of ciprofloxacin into sputum is high relative to serumconcentrations, the absolute concentrations are low relative to MICs oftarget organisms such as Pa. This data is consistant with poor clinicaloutcome due to resistance development to these low drug concentrations.

While data for levofloxacin intrapulmonary pharmacokinetics in cysticfibrosis are not available, data on the closely-related ofloxacin werepublished in the 1980's and 1990s. Ofloxacin is comprised of a racemicmixture of the dextro-(microbiologically inactive) and levo-rotatory(levofloxacin-microbiologically active). Studies have shown that thepharmacokinetic properties of the 2 components are similar. Incomparative studies with ciprofloxacin, ofloxacin had a longer half-lifeand higher distribution into sputum (79% vs 21%) than ciprofloxacin.

Lung Epithelial Lining Fluid

More recent emphasis on the use and development of fluoroquinolones incommunity acquired gram-positive infections has focused onintrapulmonary pharmacokinetic studies in lung epithelial lining fluid(ELF). Although the relevance of drug distribution into this fluid isnot clear in the setting of cystic fibrosis, insights in the drugpharmacology can be obtained from these studies. Levofloxacin penetrateswell into lung tissues. Lung tissue concentrations are generally 2- to5-fold higher than plasma concentrations. Several recent studies(summarized in Table 1) demonstrated that ELF concentrations oflevofloxacin in healthy subjects following an oral dose of 750 mg reacha maximum concentration around 20 μg/mL. Similar peak concentrations areexpected in the sputum of CF patients after oral or IV administration of750 mg of levofloxacin. In contrast, ciprofloxacin penetrates lungtissues much less efficiently than levofloxacin. Based upon studies ofthe mutant selection window (MSW), these ELF fluoroquinolone drug levelsare insufficient to achieve the required mutant prevention concentrationof 10- to 20-fold the MIC for the infecting organism.

TABLE 1 Concentration of Levofloxacin in Epithelial Linking Fluid in ManELF Drug Concentration (μg/ml) Drug Dose Route 0.5 hr 1 hr 2 hr 4 hr 6hr 12 hr 24 hr levofloxacin 500 mg IV 11 2.5 1.24 levofloxacin 500 mgoral 9.9 6.5 0.7 levofloxacin 500 mg oral 9.94 6.46 0.7 levofloxacin 500mg oral 4.74 10.8 9 10.9 9.6 levofloxacin 750 mg IV 12.94 6.04 1.73levofloxacin 750 mg oral 22.1 9.2 1.5 levofloxacin 750 mg oral 22.139.19 1.55 ciprofloxacin 500 mg oral 1.9 0.4

Quinolones

Non-limiting examples of quinolones for use as described herein includeamifloxacin, cinoxacin, ciprofloxacin, enoxacin, fleroxacin, flumequine,lomefloxacin, nalidixic acid, norfloxacin, ofloxacin, levofloxacin,lomefloxacin, oxolinic acid, pefloxacin, rosoxacin, temafloxacin,tosufloxacin, sparfloxacin, clinafloxacin, gatifloxacin, moxifloxacin;gemifloxacin; garenoxacin; olamufloxacin, clinofloxacin, trovafloxacin,balofloxacin, prulifloxacin, moxifloxacin, gemifloxacin, rufloxacin,sitafloxacin (Sato, K, et al., 1992, Antimicrob Agents Chemother.37:1491-98, which is incorporated herein by reference in its entirety),marbofloxacin, orbifloxacin, sarafloxacin, danofloxacin, difloxacin,enrofloxacin,TG-873870, DX-619, DW-276, ABT-492, DV-7751a (Tanaka, M, etal., 1992, Antimicrob. Agents Chemother. 37:2212-18), and F-1061(Kurosaka et al., Interscience Conference on Antimicrobial Agents andChemotherapy, 2003, 43rd:Chicago, which is incorporated herein byreference in its entirety).

Methods of Treatment or Prophylaxis

In some embodiments, a method is provided for treating a microbialinfection in an animal, specifically including in a mammal, by treatingan animal suffering from such an infection with a fluoroquinoloneantimicrobial. In some embodiments, fluoroquinolone antimicrobials maybe administered following aerosol formation and inhalation. Thus, thismethod of treatment is especially appropriate for the treatment ofpulmonary infections involving microbial strains that are difficult totreat using an antimicrobial agent delivered parenterally due to theneed for high parenteral dose levels (which can cause undesirable sideeffects), or due to lack of any clinically effective antimicrobialagents. In one such embodiment, this method may be used to administer afluoroquinolone antimicrobial directly to the site of infection. Such amethod may reduce systemic exposure and maximizes the amount ofantimicrobial agent to the site of microbial infection. This method isalso appropriate for treating infections involving microbes that aresusceptible to fluoroquinolone antimicrobials as a way of reducing thefrequency of selection of resistant microbes. This method is alsoappropriate for treating infections involving microbes that areotherwise resistant to fluoroquinolone antimicrobials as a way ofincreasing the amount of antimicrobial at the site of microbialinfection. A subject may be identified as infected with bacteria thatare capable of developing resistance by diagnosing the subject as havingsymptoms that are characteristic of a bacterial infection with abacteria species known to have resistant strains or a with a bacteriathat is a member of group that are known to have resistant strains.Alternatively, the bacteria may be cultured and identified as a speciesknown to have resistant strains or a bacteria that is a member of groupthat are known to have resistant strains.

In some embodiments, the aerosol fluoroquinolone antimicrobial agent isadministered at a level sufficient to overcome the emergence resistancein bacteria or increase killing efficiency such that resistance does nothave the opportunity to develop.

In some embodiments, the aerosol fluoroquinolone therapy may beadministered as a treatment or prophylaxis in combination or alternatingtherapeutic sequence with other aerosol, oral or parenteral antibiotics.By non-limiting example this may include aerosol tobramycin and/or otheraminoglycoside, aerosol aztreonam and/or other beta or mono-bactam,aerosol ciprofloxacin and/or other fluoroquinolones, aerosolazithromycin and/or other macrolides or ketolides, tetracycline and/orother tetracyclines, quinupristin and/or other streptogramins, linezolidand/or other oxazolidinones, vancomycin and/or other glycopeptides, andchloramphenicol and/or other phenicols, and colisitin and/or otherpolymyxins.

Pharmaceutical Compositions

For purposes of the method described herein, a fluoroquinoloneantimicrobial agent may be administered using an inhaler. In someembodiments, a fluoroquinolone antimicrobial disclosed herein isproduced as a pharmaceutical composition suitable for aerosol formation,good taste, storage stability, and patient safety and tolerability.

In some embodiments, the isoform content of the manufacturedfluoroquinolone may be optimized for tolerability, antimicrobialactivity and stability.

Administration

The fluoroquinolone antimicrobials disclosed herein can be administeredat a therapeutically effective dosage, e.g., a dosage sufficient toprovide treatment for the disease states previously described. Whileoptimum human dosage levels have yet to be determined for aerosoldelivery, generally a daily aerosol dose of levofloxacin (and for mostfluoroquinolone antimicrobial agents described herein) is from about 0.1to 10 mg/kg of body weight, preferably about 0.20 to 5.0 mg/kg of bodyweight, and most preferably about 0.4 to 4.0 mg/kg of body weight. Thus,for administration to a 70 kg person, the dosage range would be about7.0 to 700.0 mg per day, preferably about 14.0 to 350.0 mg per day, andmost preferably about 28.0 to 280.0 mg per day. The amount of activecompound administered will, of course, be dependent on the subject anddisease state being treated, the severity of the affliction, the mannerand schedule of administration, and the judgment of the prescribingphysician; for example, a likely dose range for aerosol administrationof levofloxacin would be about 20 to 400 mg per day.

Administration of the fluoroquinolone antimicrobial agents disclosedherein or the pharmaceutically acceptable salts thereof can be via anyof the accepted modes of administration for agents that serve similarutilities including, but not limited to, aerosol inhalation.

Pharmaceutically acceptable compositions include solid, semi-solid,liquid and aerosol dosage forms, such as, e.g., powders, liquids,suspensions, complexations, liposomes, particulates, or the like.Preferably, the compositions are provided in unit dosage forms suitablefor single administration of a precise dose. The unit dosage form canalso be assembled and packaged together to provide a patient with aweekly or monthly supply and can also incorporate other compounds suchas saline, taste masking agents, pharmaceutical excipients, and otheractive ingredients or carriers.

The fluoroquinolone antimicrobial agent can be administered either aloneor more typically in combination with a conventional pharmaceuticalcarrier, excipient or the like (e.g., mannitol, lactose, starch,magnesium stearate, sodium saccharine, talcum, cellulose, sodiumcrosscarmellose, glucose, gelatin, sucrose, magnesium carbonate,magnesium chloride, magnesium sulfate, calcium chloride, lactose,sucrose, glucose and the like). If desired, the pharmaceuticalcomposition can also contain minor amounts of nontoxic auxiliarysubstances such as wetting agents, emulsifying agents, solubilizingagents, pH buffering agents and the like (e.g., sodium acetate, sodiumcitrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamineacetate, triethanolamine oleate, and the like). Generally, depending onthe intended mode of administration, the pharmaceutical formulation willcontain about 0.005% to 95%, preferably about 0.5% to 50% by weight of acompound of the invention. Actual methods of preparing such dosage formsare known, or will be apparent, to those skilled in this art; forexample, see Remington's Pharmaceutical Sciences, Mack PublishingCompany, Easton, Pa.

In one preferred embodiment, the compositions will take the form of aunit dosage form such as vial containing a liquid, solid to besuspended, dry powder, lyophilisate, or other composition and thus thecomposition may contain, along with the active ingredient, a diluentsuch as lactose, sucrose, dicalcium phosphate, or the like; a lubricantsuch as magnesium stearate or the like; and a binder such as starch, gumacacia, polyvinylpyrrolidine, gelatin, cellulose, cellulose derivativesor the like.

Liquid pharmaceutically administrable compositions can, for example, beprepared by dissolving, dispersing, etc. an active compound as definedabove and optional pharmaceutical adjuvants in a carrier (e.g., water,saline, aqueous dextrose, glycerol, glycols, ethanol or the like) toform a solution or suspension. Solutions to be aerosolized can beprepared in conventional forms, either as liquid solutions orsuspensions, as emulsions, or in solid forms suitable for dissolution orsuspension in liquid prior to aerosol production and inhalation. Thepercentage of active compound contained in such aerosol compositions ishighly dependent on the specific nature thereof, as well as the activityof the compound and the needs of the subject. However, percentages ofactive ingredient of 0.01% to 90% in solution are employable, and willbe higher if the composition is a solid, which will be subsequentlydiluted to the above percentages. In some embodiments, the compositionwill comprise 1.0%-50.0% of the active agent in solution.

Fluoroquinolone formulations can be separated into two groups; those ofsimple formulation and complex formulations providing taste-maskingproperties, improved tolerability and/or an AUC shape-enhancingformulation. Simple formulations can be further separated into threegroups. 1. Simple formulations may include water-based liquidformulations for nebulization. By non-limiting example water-basedliquid formulations may consist of fluoroquinolone alone or withnon-encapsulating water soluble excipients. 2. Simple formulations mayalso include organic-based liquid formulations for nebulization ormeter-dose inhaler. By non-limiting example organic-based liquidformulations may consist of fluoroquinolone or with non-encapsulatingorganic soluble excipients. 3. Simple formulations may also include drypowder formulations for administration with a dry powder inhaler. Bynon-limiting example dry powder formulations may consist offluoroquinolone alone or with either water soluble or organic solublenon-encapsulating excipients with or without a blending agent such aslactose. Complex formulations can be further separated into fivegroups. 1. Complex formulations may include water-based liquidformulations for nebulization. By non-limiting example water-basedliquid complex formulations may consist of fluoroquinolone encapsulatedor complexed with water-soluble excipients such as lipids, liposomes,cyclodextrins, microencapsulations, and emulsions. 2. Complexformulations may also include organic-based liquid formulations fornebulization or meter-dose inhaler. By non-limiting exampleorganic-based liquid complex formulations may consist of fluoroquinoloneencapsulated or complexed with organic-soluble excipients such aslipids, microencapsulations, and reverse-phase water-based emulsions. 3.Complex formulations may also include low-solubility, water-based liquidformulations for nebulization. By non-limiting example low-solubility,water-based liquid complex formulations may consist of fluoroquinoloneas a low-water soluble, stable nanosuspension alone or inco-crystal/co-precipitate excipient complexes, or mixtures with lowsolubility lipids, such as lipid nanosuspensions. 4. Complexformulations may also include low-solubility, organic-based liquidformulations for nebulization or meter-dose inhaler. By non-limitingexample low-solubility, organic-based liquid complex formulations mayconsist of fluoroquinolone as a low-organic soluble, stablenanosuspension alone or in co-crystal/co-precipitate excipientcomplexes, or mixtures with low solubility lipids, such as lipidnanosuspensions. 5. Complex formulations may also include dry powderformulations for administration using a dry powder inhaler. Bynon-limiting example, complex dry powder formulations may consist offluoroquinolone in co-crystal/co-precipitate/spray dried complex ormixture with low-water soluble excipients/salts in dry powder form withor without a blending agent such as lactose. Specific methods for simpleand complex formulation preparation are described herein.

Aerosol Delivery

Fluoroquinolone antimicrobial agents as described herein, are preferablydirectly administered as an aerosol to a site of infection in therespiratory tract. In some embodiments, aerosol delivery is used totreat an infection in the lungs, such as a Pseudomonas lung infection.

Several device technologies exist to deliver either dry powder or liquidaerosolized products. Dry powder formulations generally require lesstime for drug administration, yet longer and more expensive developmentefforts. Conversely, liquid formulations have historically suffered fromlonger administration times, yet have the advantage of shorter and lessexpensive development efforts. The fluoroquinolone antimicrobial agentsdisclosed herein range in solubility, are generally stable and have arange of tastes. In one such embodiment, the fluoroquinoloneantimicrobial levofloxacin is water soluble at neutral pH, is stable inaqueous solution and has limited to no taste.

Accordingly, in one embodiment, a particular formulation offluoroquinolone antimicrobial agent disclosed herein is combined with aparticular aerosolizing device to provide an aerosol for inhalation thatis optimized for maximum drug deposition at a site of infection andmaximal tolerability. Factors that can be optimized include solution orsolid particle formulation, rate of delivery, and particle size anddistribution produced by the aerosolizing device.

Particle Size and Distribution

Generally, inhaled particles are subject to deposition by one of twomechanisms: impaction, which usually predominates for larger particles,and sedimentation, which is prevalent for smaller particles. Impactionoccurs when the momentum of an inhaled particle is large enough that theparticle does not follow the air stream and encounters a physiologicalsurface. In contrast, sedimentation occurs primarily in the deep lungwhen very small particles which have traveled with the inhaled airstream encounter physiological surfaces as a result of random diffusionwithin the air stream.

For pulmonary administration, the upper airways are avoided in favor ofthe middle and lower airways. Pulmonary drug delivery may beaccomplished by inhalation of an aerosol through the mouth and throat.Particles having a mass median aerodynamic diameter (MMAD) of greaterthan about 5 microns generally do not reach the lung; instead, they tendto impact the back of the throat and are swallowed and possibly orallyabsorbed. Particles having diameters of about 2 to about 5 microns aresmall enough to reach the upper- to mid-pulmonary region (conductingairways), but are too large to reach the alveoli. Smaller particles,i.e., about 0.5 to about 2 microns, are capable of reaching the alveolarregion. Particles having diameters smaller than about 0.5 microns canalso be deposited in the alveolar region by sedimentation, although verysmall particles may be exhaled. Measures of particle size can bereferred to as volumetric mean diameter (VIVID), mass median diameter(MMD), or MMAD. These measurements may be made by impaction (MMD andMMAD) or by laser (VMD). For liquid particles, VIVID, MMD and MMAD maybe the same if environmental conditions are maintained, e.g. standardhumidity. However, if humidity is not maintained, MMD and MMADdeterminations will be smaller than VIVID due to dehydration duringimpactor measurements. For the purposes of this description, VIVID, MMDand MMAD measurements are considered to be under standard conditionssuch that descriptions of VIVID, MMD and MMAD will be comparable.Similarly, dry powder particle size determinations in MMD, and MMAD arealso considered comparable.

In some embodiments, the particle size of the aerosol is optimized tomaximize fluoroquinolone antimicrobial agent deposition at the site ofinfection and to maximize tolerability. Aerosol particle size may beexpressed in terms of the mass median aerodynamic diameter (MMAD). Largeparticles (e.g., MMAD >5 μm) may deposit in the upper airway becausethey are too large to navigate the curvature of the upper airway. Smallparticles (e.g., MMAD <2 μm) may be poorly deposited in the lowerairways and thus become exhaled, providing additional opportunity forupper airway deposition. Hence, intolerability (e.g., cough andbronchospasm) may occur from upper airway deposition from bothinhalation impaction of large particles and settling of small particlesduring repeated inhalation and expiration. Thus, in one embodiment, anoptimum particle size is used (e.g., MMAD=2-5 μm) in order to maximizedeposition at a mid-lung site of infection and to minimizeintoleratiblity associated with upper airway deposition. Moreover,generation of a defined particle size with limited geometric standarddeviation (GSD) may optimize deposition and tolerability. Narrow GSDlimits the number of particles outside the desired MMAD size range. Inone embodiment, an aerosol containing one or more compounds disclosedherein is provided having a MMAD from about 2 microns to about 5 micronswith a GSD of less than or equal to about 2.5 microns. In anotherembodiment, an aerosol having an MMAD from about 2.8 microns to about4.3 microns with a GSD less than or equal to 2 microns is provided. Inanother embodiment, an aerosol having an MMAD from about 2.5 microns toabout 4.5 microns with a GSD less than or equal to 1.8 microns isprovided.

Fluoroquinolone antimicrobial agents disclosed herein intended forrespiratory delivery (for either systemic or local distribution) can beadministered as aqueous formulations, as suspensions or solutions inhalogenated hydrocarbon propellants, or as dry powders. Aqueousformulations may be aerosolized by liquid nebulizers employing eitherhydraulic or ultrasonic atomization. Propellant-based systems may usesuitable pressurized metered-dose inhalers (pMDIs). Dry powders may usedry powder inhaler devices (DPIs), which are capable of dispersing thedrug substance effectively. A desired particle size and distribution maybe obtained by choosing an appropriate device.

Liquid Nebulizer

In one embodiment, a nebulizer is selected on the basis of allowing theformation of an aerosol of a fluoroquinolone antimicrobial agentdisclosed herein having an MMAD predominantly between about 2 to about 5microns. In one embodiment, the delivered amount of fluoroquinoloneantimicrobial agent provides a therapeutic effect for respiratoryinfections.

Previously, two types of nebulizers, jet and ultrasonic, have been shownto be able to produce and deliver aerosol particles having sizes between2 and 4 um. These particle sizes have been shown as being optimal fortreatment of pulmonary bacterial infection cause by gram-negativebacteria such as Pseudomonas aeruginosa, Escherichia coli, Enterobacterspecies, Klebsiella pneumoniae, K. oxytoca, Proteus mirabilis,Pseudomonas aeruginosa, Serratia marcescens, Haemophilus influenzae,Burkholderia cepacia, Stenotrophomonas maltophilia, Alcaligenesxylosoxidans, and multidrug resistant Pseudomonas aeruginosa. However,unless a specially formulated solution is used, these nebulizerstypically need larger volumes to administer sufficient amount of drug toobtain a therapeutic effect. A jet nebulizer utilizes air pressurebreakage of an aqueous solution into aerosol droplets. An ultrasonicnebulizer utilizes shearing of the aqueous solution by a piezoelectriccrystal. Typically, however, the jet nebulizers are only about 10%efficient under clinical conditions, while the ultrasonic nebulizer isonly about 5% efficient. The amount of pharmaceutical deposited andabsorbed in the lungs is thus a fraction of the 10% in spite of thelarge amounts of the drug placed in the nebulizer.

Accordingly, in one embodiment, a vibrating mesh nebulizer is used todeliver an aerosol of the fluoroquinolone antimicrobial agent disclosedherein. A vibrating mesh nebulizer consists of a liquid storagecontainer in fluid contact with a diaphragm and inhalation andexhalation valves. In one embodiment, about 1 to about 5 ml of thefluoroquinolone antimicrobial agent is placed in the storage containerand the aerosol generator is engaged producing atomized aerosol ofparticle sizes selectively between about 1 and about 5 um.

By non-limiting example, a fluoroquinolone antimicrobial agent disclosedherein is placed in a liquid nebulization inhaler and prepared indosages to deliver from about 7 to about 700 mg from a dosing solutionof about 1 to about 5 ml, preferably from about 14 to about 350 mg inabout 1 to about 5 ml, and most preferably from about 28 to about 280 mgin about 1 to about 5 ml with MMAD particles sizes between about 2 toabout 5 um being produced.

By non-limiting example, a nebulized fluoroquinolone antimicrobial maybe administered in the described respirable delivered dose in less thanabout 20 min, preferably less than about 10 min, more preferably lessthan about 7 min, more preferably less than about 5 min, more preferablyless than about 3 min, and in some cases most preferable if less thanabout 2 min.

By non-limiting example, in other circumstances, a nebulizedfluoroquinolone antimicrobial may achieve improved tolerability and/orexhibit an AUC shape-enhancing characteristic when administered overlonger periods of time. Under these conditions, the described respirabledelivered dose in more than about 2 min, preferably more than about 3min, more preferably more than about 5 min, more preferably more thanabout 7 min, more preferably more than about 10 min, and in some casesmost preferable from about 10 to about 20 min.

For aqueous and other non-pressurized liquid systems, a variety ofnebulizers (including small volume nebulizers) are available toaerosolize the formulations. Compressor-driven nebulizers incorporatejet technology and use compressed air to generate the liquid aerosol.Such devices are commercially available from, for example, HealthdyneTechnologies, Inc.; Invacare, Inc.; Mountain Medical Equipment, Inc.;Pari Respiratory, Inc.; Mada Medical, Inc.; Puritan-Bennet; Schuco,Inc., DeVilbiss Health Care, Inc.; and Hospitak, Inc. Ultrasonicnebulizers rely on mechanical energy in the form of vibration of apiezoelectric crystal to generate respirable liquid droplets and arecommercially available from, for example, Omron Heathcare, Inc. andDeVilbiss Health Care, Inc. Vibrating mesh nebulizers rely upon eitherpiezoelectric or mechanical pulses to respirable liquid dropletsgenerate. Other examples of nebulizers for use with fluoroquinoloneantimicrobial agents described herein are described in U.S. Pat. Nos.4,268,460; 4,253,468; 4,046,146; 3,826,255; 4,649,911; 4,510,929;4,624,251; 5,164,740; 5,586,550; 5,758,637; 6,644,304; 6,338,443;5,906,202; 5,934,272; 5,960,792; 5,971,951; 6,070,575; 6,192,876;6,230,706; 6,349,719; 6,367,470; 6,543,442; 6,584,971; 6,601,581;4,263,907; 5,709,202; 5,823,179; 6,192,876; 6,644,304; 5,549,102;6,083,922; 6,161,536; 6,264,922; 6,557,549; and 6,612,303 all of whichare hereby incorporated by reference in their entirety. Commercialexamples of nebulizers that can be used with the fluoroquinoloneantimicrobial agents described herein include Respirgard II®, Aeroneb®,Aeroneb® Pro, and Aeroneb® Go produced by Aerogen; AERx® and AERxEssence™ produced by Aradigm; Porta-Neb®, Freeway Freedom™,Sidestream-Ventstream and I-neb produced by Respironics, Inc.; and PAMLC-Plus®, PAM LC-Star®, and e-Flow^(7m) produced by PAM, GmbH. Byfurther non-limiting example, U.S. Pat. No. 6,196,219, is herebyincorporated by reference in its entirety.

In some embodiments, the drug solution is formed prior to use of thenebulizer by a patient. In other embodiments, the drug is stored in thenebulizer in solid form. In this case, the solution is mixed uponactivation of the nebulizer, such as described in U.S. Pat. No.6,427,682 and PCT Publication No. WO 03/035030, both of which are herebyincorporated by reference in their entirety. In these nebulizers, thesolid drug, optionally combined with excipients to form a solidcomposition, is stored in a separate compartment from a liquid solvent.

The liquid solvent is capable of dissolving the solid composition toform a liquid composition, which can be aerosolized and inhaled. Suchcapability is, among other factors, a function of the selected amountand, potentially, the composition of the liquid. To allow easy handlingand reproducible dosing, the sterile aqueous liquid may be able todissolve the solid composition within a short period of time, possiblyunder gentle shaking. In some embodiments, the final liquid is ready touse after no longer than about 30 seconds. In some cases, the solidcomposition is dissolved within about 20 seconds, and advantageously,within about 10 seconds. As used herein, the terms “dissolve(d)”,“dissolving”, and “dissolution” refer to the disintegration of the solidcomposition and the release, i.e. the dissolution, of the activecompound. As a result of dissolving the solid composition with theliquid solvent a liquid composition is formed in which the activecompound is contained in the dissolved state. As used herein, the activecompound is in the dissolved state when at least about 90 wt. % aredissolved, and more preferably when at least about 95 wt. % aredissolved.

With regard to basic separated-compartment nebulizer design, itprimarily depends on the specific application whether it is more usefulto accommodate the aqueous liquid and the solid composition withinseparate chambers of the same container or primary package, or whetherthey should be provided in separate containers. If separate containersare used, these are provided as a set within the same secondary package.The use of separate containers is especially preferred for nebulizerscontaining two or more doses of the active compound. There is no limitto the total number of containers provided in a multi-dose kit. In oneembodiment, the solid composition is provided as unit doses withinmultiple containers or within multiple chambers of a container, whereasthe liquid solvent is provided within one chamber or container. In thiscase, a favorable design provides the liquid in a metered-dosedispenser, which may consist of a glass or plastic bottle closed with adispensing device, such as a mechanical pump for metering the liquid.For instance, one actuation of the pumping mechanism may dispense theexact amount of liquid for dissolving one dose unit of the solidcomposition.

In another embodiment for multiple-dose separated-compartmentnebulizers, both the solid composition and the liquid solvent areprovided as matched unit doses within multiple containers or withinmultiple chambers of a container. For instance, two-chambered containerscan be used to hold one unit of the solid composition in one of thechambers and one unit of liquid in the other. As used herein, one unitis defined by the amount of drug present in the solid composition, whichis one unit dose. Such two-chambered containers may, however, also beused advantageously for nebulizers containing only one single drug dose.

In one embodiment of a separated-compartment nebulizer, a blister packhaving two blisters is used, the blisters representing the chambers forcontaining the solid composition and the liquid solvent in matchedquantities for preparing a dose unit of the final liquid composition. Asused herein, a blister pack represents a thermoformed or pressure-formedprimary packaging unit, most likely comprising a polymeric packagingmaterial that optionally includes a metal foil, such as aluminum. Theblister pack may be shaped to allow easy dispensing of the contents. Forinstance, one side of the pack may be tapered or have a tapered portionor region through which the content is dispensable into another vesselupon opening the blister pack at the tapered end. The tapered end mayrepresent a tip.

In some embodiments, the two chambers of the blister pack are connectedby a channel, the channel being adapted to direct fluid from the blistercontaining the liquid solvent to the blister containing the solidcomposition. During storage, the channel is closed with a seal. In thissense, a seal is any structure that prevents the liquid solvent fromcontacting the solid composition. The seal is preferably breakable orremovable; breaking or removing the seal when the nebulizer is to beused will allow the liquid solvent to enter the other chamber anddissolve the solid composition. The dissolution process may be improvedby shaking the blister pack. Thus, the final liquid composition forinhalation is obtained, the liquid being present in one or both of thechambers of the pack connected by the channel, depending on how the packis held.

According to another embodiment, one of the chambers, preferably the onethat is closer to the tapered portion of the blister pack, communicateswith a second channel, the channel extending from the chamber to adistal position of the tapered portion. During storage, this secondchannel does not communicate with the outside of the pack but is closedin an air-tight fashion. Optionally, the distal end of the secondchannel is closed by a breakable or removable cap or closure, which maye.g. be a twist-off cap, a break-off cap, or a cut-off cap.

In one embodiment, a vial or container having two compartments is used,the compartment representing the chambers for containing the solidcomposition and the liquid solvent in matched quantities for preparing adose unit of the final liquid composition. The liquid composition and asecond liquid solvent may be contained in matched quantities forpreparing a dose unit of the final liquid composition (by non-limitingexample in cases where two soluble excipients or the fluoroquinolone andexcipient are unstable for storage, yet desired in the same mixture foradministration.

In some embodiments, the two compartments are physically separated butin fluid communication such as when so the vial or container areconnected by a channel or breakable barrier, the channel or breakablebarrier being adapted to direct fluid between the two compartments toenable mixing prior to administration. During storage, the channel isclosed with a seal or the breakable barrier intact. In this sense, aseal is any structure that prevents mixing of contents in the twocompartments. The seal is preferably breakable or removable; breaking orremoving the seal when the nebulizer is to be used will allow the liquidsolvent to enter the other chamber and dissolve the solid composition orin the case of two liquids permit mixing. The dissolution or mixingprocess may be improved by shaking the container. Thus, the final liquidcomposition for inhalation is obtained, the liquid being present in oneor both of the chambers of the pack connected by the channel orbreakable barrier, depending on how the pack is held.

The solid composition itself can be provided in various different typesof dosage forms, depending on the physicochemical properties of thedrug, the desired dissolution rate, cost considerations, and othercriteria. In one of the embodiments, the solid composition is a singleunit. This implies that one unit dose of the drug is comprised in asingle, physically shaped solid form or article. In other words, thesolid composition is coherent, which is in contrast to a multiple unitdosage form, in which the units are incoherent.

Examples of single units which may be used as dosage forms for the solidcomposition include tablets, such as compressed tablets, film-likeunits, foil-like units, wafers, lyophilized matrix units, and the like.In a preferred embodiment, the solid composition is a highly porouslyophilized form. Such lyophilizates, sometimes also called wafers orlyophilized tablets, are particularly useful for their rapiddisintegration, which also enables the rapid dissolution of the activecompound.

On the other hand, for some applications the solid composition may alsobe formed as a multiple unit dosage form as defined above. Examples ofmultiple units are powders, granules, microparticles, pellets, beads,lyophilized powders, and the like. In one embodiment, the solidcomposition is a lyophilized powder. Such a dispersed lyophilized systemcomprises a multitude of powder particles, and due to the lyophilizationprocess used in the formation of the powder, each particle has anirregular, porous microstructure through which the powder is capable ofabsorbing water very rapidly, resulting in quick dissolution.

Another type of multiparticulate system which is also capable ofachieving rapid drug dissolution is that of powders, granules, orpellets from water-soluble excipients which are coated with the drug, sothat the drug is located at the outer surface of the individualparticles. In this type of system, the water-soluble low molecularweight excipient is useful for preparing the cores of such coatedparticles, which can be subsequently coated with a coating compositioncomprising the drug and, preferably, one or more additional excipients,such as a binder, a pore former, a saccharide, a sugar alcohol, afilm-forming polymer, a plasticizer, or other excipients used inpharmaceutical coating compositions.

In another embodiment, the solid composition resembles a coating layerthat is coated on multiple units made of insoluble material. Examples ofinsoluble units include beads made of glass, polymers, metals, andmineral salts. Again, the desired effect is primarily rapiddisintegration of the coating layer and quick drug dissolution, which isachieved by providing the solid composition in a physical form that hasa particularly high surface-to-volume ratio. Typically, the coatingcomposition will, in addition to the drug and the water-soluble lowmolecular weight excipient, comprise one or more excipients, such asthose mentioned above for coating soluble particles, or any otherexcipient known to be useful in pharmaceutical coating compositions.

To achieve the desired effects, it may be useful to incorporate morethan one water-soluble low molecular weight excipient into the solidcomposition. For instance, one excipient may be selected for its drugcarrier and diluent capability, while another excipient may be selectedto adjust the pH. If the final liquid composition needs to be buffered,two excipients that together form a buffer system may be selected.

In one embodiment, the liquid to be used in a separated-compartmentnebulizer is an aqueous liquid, which is herein defined as a liquidwhose major component is water. The liquid does not necessarily consistof water only; however, in one embodiment it is purified water. Inanother embodiment, the liquid contains other components or substances,preferably other liquid components, but possibly also dissolved solids.Liquid components other than water which may be useful include propyleneglycol, glycerol, and polyethylene glycol. One of the reasons toincorporate a solid compound as a solute is that such a compound isdesirable in the final liquid composition, but is incompatible with thesolid composition or with a component thereof, such as the activeingredient.

Another desirable characteristic for the liquid solvent is that it issterile. An aqueous liquid would be subject to the risk of considerablemicrobiological contamination and growth if no measures were taken toensure sterility. In order to provide a substantially sterile liquid, aneffective amount of an acceptable antimicrobial agent or preservativecan be incorporated or the liquid can be sterilized prior to providingit and to seal it with an air-tight seal. In one embodiment, the liquidis a sterilized liquid free of preservatives and provided in anappropriate air-tight container. However, according to anotherembodiment in which the nebulizer contains multiple doses of the activecompound, the liquid may be supplied in a multiple-dose container, suchas a metered-dose dispenser, and may require a preservative to preventmicrobial contamination after the first use.

Meter Dose Inhaler (MDI)

A propellant driven inhaler (pMDI) releases a metered dose of medicineupon each actuation. The medicine is formulated as a suspension orsolution of a drug substance in a suitable propellant such as ahalogenated hydrocarbon. pMDIs are described in, for example, Newman, S.P., Aerosols and the Lung, Clarke et al., eds., pp. 197-224(Butterworths, London, England, 1984).

In some embodiments, the particle size of the drug substance in an MDImay be optimally chosen. In some embodiments, the particles of activeingredient have diameters of less than about 50 microns. In someembodiments, the particles have diameters of less than about 10 microns.In some embodiments, the particles have diameters of from about 1 micronto about 5 microns. In some embodiments, the particles have diameters ofless than about 1 micron. In one advantageous embodiment, the particleshave diameters of from about 2 microns to about 5 microns.

The propellants for use with the MDIs may be any propellants known inthe art. Examples of propellants include chlorofluorocarbons (CFCs) suchas dichlorodifluoromethane, trichlorofluorometbane, anddichlorotetrafluoroethane; hydrofluoroalkanes (HFAs); and carbondioxide. It may be advantageous to use HFAs instead of CFCs due to theenvironmental concerns associated with the use of CFCs. Examples ofmedicinal aerosol preparations containing HFAs are presented in U.S.Pat. Nos. 6,585,958; 2,868,691 and 3,014,844, all of which are herebyincorporated by reference in their entirety. In some embodiments, aco-solvent is mixed with the propellant to facilitate dissolution orsuspension of the drug substance.

In some embodiments, the propellant and active ingredient are containedin separate containers, such as described in U.S. Pat. No. 4,534,345,which is hereby incorporated by reference in its entirety.

In some embodiments, the MDI used herein is activated by a patientpushing a lever, button, or other actuator. In other embodiments, therelease of the aerosol is breath activated such that, after initiallyarming the unit, the active compound aerosol is released once thepatient begins to inhale, such as described in U.S. Pat. Nos. 6,672,304;5,404,871; 5,347,998; 5,284,133; 5,217,004; 5,119,806; 5,060,643;4,664,107; 4,648,393; 3,789,843; 3,732,864; 3,636,949; 3,598,294;3,565,070; 3,456,646; 3,456,645; and 3,456,644, each of which is herebyincorporated by reference in its entirety. Such a system enables more ofthe active compound to get into the lungs of the patient. Anothermechanism to help a patient get adequate dosage with the activeingredient may include a valve mechanism that allows a patient to usemore than one breath to inhale the drug, such as described in U.S. Pat.Nos. 4,470,412 and 5,385,140, both of which are hereby incorporated byreference in their entirety.

Additional examples of MDIs known in the art and suitable for use hereininclude U.S. Pat. Nos. 6,435,177; 6,585,958; 5,642,730; 6,223,746;4,955,371; 5,404,871; 5,364,838; and 6,523,536, all of which are herebyincorporated by reference in their entirety.

Dry Powder Inhaler (DPI)

There are two major designs of dry powder inhalers. One design is themetering device in which a reservoir for the drug is placed within thedevice and the patient adds a dose of the drug into the inhalationchamber. The second is a factory-metered device in which each individualdose has been manufactured in a separate container. Both systems dependupon the formulation of drug into small particles of mass mediandiameters from about 1 to about 5 um, and usually involve co-formulationwith larger excipient particles (typically 100 um diameter lactoseparticles). Drug powder is placed into the inhalation chamber (either bydevice metering or by breakage of a factory-metered dosage) and theinspiratory flow of the patient accelerates the powder out of the deviceand into the oral cavity. Non-laminar flow characteristics of the powderpath cause the excipient-drug aggregates to decompose, and the mass ofthe large excipient particles causes their impaction at the back of thethroat, while the smaller drug particles are deposited deep in thelungs.

As with liquid nebulization and MDIs, particle size of thefluoroquinolone antimicrobial agent aerosol formulation may beoptimized. If the particle size is larger than about 5 um MMAD then theparticles are deposited in upper airways. If the particle size of theaerosol is smaller than about 1 um then it is delivered into the alveoliand may get transferred into the systemic blood circulation.

By non-limiting example, in dry powder inhalers, the fluoroquinoloneantimicrobial agents disclosed herein are prepared in dosages to deliverfrom about 7 to about 700 mg from a dosing solution of about 1 to about5 ml, preferably from about 14 to about 350 mg in about 1 to about 5 ml,and most preferably from about 28 to about 280 mg in about 1 to about 5ml with MMAD particles sizes between about 2 to about 5 um beingproduced.

In some embodiments, a dry powder inhaler (DPI) is used to dispense thefluoroquinolone antimicrobial agents described herein. DPIs contain thedrug substance in fine dry particle form. Typically, inhalation by apatient causes the dry particles to form an aerosol cloud that is drawninto the patient's lungs. The fine dry drug particles may be produced byany technique known in the art. Some well-known techniques include useof a jet mill or other comminution equipment, precipitation fromsaturated or super saturated solutions, spray drying, in situmicronization (Hovione), or supercritical fluid methods. Typical powderformulations include production of spherical pellets or adhesivemixtures. In adhesive mixtures, the drug particles are attached tolarger carrier particles, such as lactose monohydrate of size about 50to about 100 microns in diameter. The larger carrier particles increasethe aerodynamic forces on the carrier/drug agglomerates to improveaerosol formation. Turbulence and/or mechanical devices break theagglomerates into their constituent parts. The smaller drug particlesare then drawn into the lungs while the larger carrier particles depositin the mouth or throat. Some examples of adhesive mixtures are describedin U.S. Pat. No. 5,478,578 and PCT Publication Nos. WO 95/11666, WO87/05213, WO 96/23485, and WO 97/03649, all of which are incorporated byreference in their entirety. Additional excipients may also be includedwith the drug substance.

There are three common types of DPIs, all of which may be used with thefluoroquinolone antimicrobial agents described herein. In a single-doseDPI, a capsule containing one dose of dry drug substance/excipients isloaded into the inhaler. Upon activation, the capsule is breached,allowing the dry powder to be dispersed and inhaled using a dry powderinhaler. To dispense additional doses, the old capsule must be removedand an additional capsule loaded. Examples of single-dose DPIs aredescribed in U.S. Pat. Nos. 3,807,400; 3,906,950; 3,991,761; and4,013,075, all of which are hereby incorporated by reference in theirentirety. In a multiple unit dose DPI, a package containing multiplesingle dose compartments is provided. For example, the package maycomprise a blister pack, where each blister compartment contains onedose. Each dose can be dispensed upon breach of a blister compartment.Any of several arrangements of compartments in the package can be used.For example, rotary or strip arrangements are common. Examples ofmultiple unit does DPIs are described in EPO Patent ApplicationPublication Nos. 0211595A2, 0455463A1, and 0467172A1, all of which arehereby incorporated by reference in their entirety. In a multi-dose DPI,a single reservoir of dry powder is used. Mechanisms are provided thatmeasure out single dose amounts from the reservoir to be aerosolized andinhaled, such as described in U.S. Pat. Nos. 5,829,434; 5,437,270;2,587,215; 5,113,855; 5,840,279; 4,688,218; 4,667,668; 5,033,463; and4,805,811 and PCT Publication No. WO 92/09322, all of which are herebyincorporated by reference in their entirety.

In some embodiments, auxiliary energy in addition to or other than apatient's inhalation may be provided to facilitate operation of a DPI.For example, pressurized air may be provided to aid in powderde-agglomeration, such as described in U.S. Pat. Nos. 3,906,950;5,113,855; 5,388,572; 6,029,662 and PCT Publication Nos. WO 93/12831, WO90/07351, and WO 99/62495, all of which are hereby incorporated byreference in their entirety. Electrically driven impellers may also beprovided, such as described in U.S. Pat. Nos. 3,948,264; 3,971,377;4,147,166; 6,006,747 and PCT Publication No. WO 98/03217, all of whichare hereby incorporated by reference in their entirety. Anothermechanism is an electrically powered tapping piston, such as describedin PCT Publication No. WO 90/13327, which is hereby incorporated byreference in its entirety. Other DPIs use a vibrator, such as describedin U.S. Pat. Nos. 5,694,920 and 6,026,809, both of which are herebyincorporated by reference in their entirety. Finally, a scraper systemmay be employed, such as described in PCT Publication No. WO 93/24165,which is hereby incorporated by reference in its entirety.

Additional examples of DPIs for use herein are described in U.S. Pat.Nos. 4,811,731; 5,113,855; 5,840,279; 3,507,277; 3,669,113; 3,635,219;3,991,761; 4,353,365; 4,889,144, 4,907,538; 5,829,434; 6,681,768;6,561,186; 5,918,594; 6,003,512; 5,775,320; 5,740,794; and 6,626,173,all of which are hereby incorporated by reference in their entirety.

In some embodiments, a spacer or chamber may be used with any of theinhalers described herein to increase the amount of drug substance thatgets absorbed by the patient, such as is described in U.S. Pat. Nos.4,470,412; 4,790,305; 4,926,852; 5,012,803; 5,040,527; 5,024,467;5,816,240; 5,027,806; and 6,026,807, all of which are herebyincorporated by reference in their entirety. For example, a spacer maydelay the time from aerosol production to the time when the aerosolenters a patient's mouth. Such a delay may improve synchronizationbetween the patient's inhalation and the aerosol production. A mask mayalso be incorporated for infants or other patients that have difficultyusing the traditional mouthpiece, such as is described in U.S. Pat. Nos.4,809,692; 4,832,015; 5,012,804; 5,427,089; 5,645,049; and 5,988,160,all of which are hereby incorporated by reference in their entirety.

Dry powder inhalers (DPIs), which involve deaggregation andaerosolization of dry powders, normally rely upon a burst of inspiredair that is drawn through the unit to deliver a drug dosage. Suchdevices are described in, for example, U.S. Pat. No. 4,807,814, which isdirected to a pneumatic powder ejector having a suction stage and aninjection stage; SU 628930 (Abstract), describing a hand-held powderdisperser having an axial air flow tube; Fox et al., Powder and BulkEngineering, pages 33-36 (March 1988), describing a venturi eductorhaving an axial air inlet tube upstream of a venturi restriction; EP 347779, describing a hand-held powder disperser having a collapsibleexpansion chamber, and U.S. Pat. No. 5,785,049, directed to dry powderdelivery devices for drugs.

Solution/Dispersion Formulations

In one embodiment, aqueous formulations containing soluble ornanoparticulate drug particles are provided. For aqueous aerosolformulations, the drug may be present at a concentration of about 1mg/mL up to about 700 mg/mL. Such formulations provide effectivedelivery to appropriate areas of the lung, with the more concentratedaerosol formulations having the additional advantage of enabling largequantities of drug substance to be delivered to the lung in a very shortperiod of time. In one embodiment, a formulation is optimized to providea well tolerated formulation. Accordingly, in one embodiment,fluoroquinolone antimicrobial agents disclosed herein are formulated tohave good taste, pH from about 5.5 to about 7, osmolarity from about 200to about 1250 mOsmol/kg, permeant ion concentration from about 30 toabout 300 mM.

In one embodiment, the solution or diluent used for preparation ofaerosol formulations has a pH range from about 4.5 to about 7.5,preferably from about 5.5 to about 7.0. This pH range improvestolerability. When the aerosol is either acidic or basic, it can causebronchospasm and cough. Although the safe range of pH is relative andsome patients may tolerate a mildly acidic aerosol, while others willexperience bronchospasm. Any aerosol with a pH of less than about 4.5typically induces bronchospasm. Aerosols with a pH from about 4.5 toabout 5.5 will cause bronchospasm occasionally. Any aerosol having pHgreater than about 7.5 may have low tolerability because body tissuesare generally unable to buffer alkaline aerosols. Aerosols withcontrolled pH below about 4.5 and over about 7.5 typically result inlung irritation accompanied by severe bronchospasm cough andinflammatory reactions. For these reasons as well as for the avoidanceof bronchospasm, cough or inflammation in patients, the optimum pH forthe aerosol formulation was determined to be between about pH5.5 toabout pH 7.0. Consequently, in one embodiment, aerosol formulations foruse as described herein are adjusted to pH between about 4.5 and about7.5 with preferred pH range from about about 5.5 to about 7.5. Mostpreferred pH range is from about 5.5 to about 7.5.

By non-limiting example, compositions may also include a buffer or a pHadjusting agent, typically a salt prepared from an organic acid or base.Representative buffers include organic acid salts of citric acid,ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinicacid, acetic acid, or phthalic acid, Tris, tromethamine, hydrochloride,or phosphate buffers.

Many patients have increased sensitivity to various chemical tastes,including bitter, salt, sweet, metallic sensations. To createwell-tolerated drug products, by non-limiting example taste masking maybe accomplished through the addition of taste-masking excipients,adjusted osmolality, and sweeteners.

Many patients have increased sensitivity to various chemical agents andhave high incidence of bronchospastic, asthmatic or other coughingincidents. Their airways are particularly sensitive to hypotonic orhypertonic and acidic or alkaline conditions and to the presence of anypermanent ion, such as chloride. Any imbalance in these conditions or apresence of chloride above certain value leads to bronchospastic orinflammatory events and/or cough which greatly impair treatment withinhalable formulations. Both these conditions prevent efficient deliveryof aerosolized drugs into the endobronchial space.

In some embodiments, the osmolality of aqueous solutions of thefluoroquinolone antimicrobial agent disclosed herein are adjusted byproviding excipients. In some cases, a certain amount of chloride oranother anion is needed for successful and efficacious delivery ofaerosolized antibiotic. However, it has been discovered that suchamounts are lower than amounts provided and typically used for aerosolsof other compounds.

Bronchospasm or cough reflexes do not respond to the same osmolality ofthe diluent for aerosolization. However, they can be sufficientlycontrolled and/or suppressed when the osmolality of the diluent is in acertain range. A preferred solution for aerosolization of therapeuticcompounds which is safe and tolerated has a total osmolality from about200 to about 1250 mOsmol/kg with a range of chloride concentration offrom about 30 mM to about 300 mM and preferably from about 50 mM toabout 150 mM. This osmolality controls bronchospasm, the chlorideconcentration, as a permeant anion, controls cough. Because they areboth permeant ions, both bromine or iodine anions may be substituted forchloride. In addition, bicarbonate may substituted for chloride ion.

By non-limiting example, the formulation for an aerosol fluoroquinoloneantimicrobial agent may comprise from about 7 to about 700 mg,preferably from about 14 to about 300 mg, or most preferably from about28 to about 280 mg fluoroquinolone antimicrobial agent per about 1 toabout 5 ml of dilute saline (between 1/10 to 1/1 normal saline).Accordingly, the concentration of a levofloxacin solution may be greaterthan about 25 mg/ml, greater than about 35 mg/ml and is preferablygreater than about 40 mg/ml, and is as high or greater than 50/ml.

In one embodiment, solution osmolality is from about 100 mOsmol/kg toabout 600 mOsmol/kg. In various other embodiments, the solutionosmolality is from about 2000 mOsmol/kg to about 1250 mOsmol/kg; fromabout 250 mOsmol/kg to about 1050 mOsmol/kg; and from about 350mOsmol/kg to about 750 mOsmol/kg.

In one embodiments, permeant ion concentration is from about 25 mM toabout 400 mM. In various other embodiments, permeant ion concentrationis from about 30 mM to about 300 mM; from about 40 mM to about 200 mM;and from about 50 mM to about 150 mM.

Solid Particle Formulations

In some embodiments, solid drug nanoparticles are provided for use ingenerating dry aerosols or for generating nanoparticles in liquidsuspension. Powders comprising nanoparticulate drug can be made byspray-drying aqueous dispersions of a nanoparticulate drug and a surfacemodifier to form a dry powder which consists of aggregated drugnanoparticles. In one embodiment, the aggregates can have a size ofabout 1 to about 2 microns which is suitable for deep lung delivery. Theaggregate particle size can be increased to target alternative deliverysites, such as the upper bronchial region or nasal mucosa by increasingthe concentration of drug in the spray-dried dispersion or by increasingthe droplet size generated by the spray dryer.

Alternatively, an aqueous dispersion of drug and surface modifier cancontain a dissolved diluent such as lactose or mannitol which, whenspray dried, forms respirable diluent particles, each of which containsat least one embedded drug nanoparticle and surface modifier. Thediluent particles with embedded drug can have a particle size of about 1to about 2 microns, suitable for deep lung delivery. In addition, thediluent particle size can be increased to target alternate deliverysites, such as the upper bronchial region or nasal mucosa by increasingthe concentration of dissolved diluent in the aqueous dispersion priorto spray drying, or by increasing the droplet size generated by thespray dryer.

Spray-dried powders can be used in DPIs or pMDIs, either alone orcombined with freeze-dried nanoparticulate powder. In addition,spray-dried powders containing drug nanoparticles can be reconstitutedand used in either jet or ultrasonic nebulizers to generate aqueousdispersions having respirable droplet sizes, where each droplet containsat least one drug nanoparticle. Concentrated nanoparticulate dispersionsmay also be used in these aspects of the invention.

Nanoparticulate drug dispersions can also be freeze-dried to obtainpowders suitable for nasal or pulmonary delivery. Such powders maycontain aggregated nanoparticulate drug particles having a surfacemodifier. Such aggregates may have sizes within a respirable range,e.g., about 2 to about 5 microns MMAD.

Freeze dried powders of the appropriate particle size can also beobtained by freeze drying aqueous dispersions of drug and surfacemodifier, which additionally contain a dissolved diluent such as lactoseor mannitol. In these instances the freeze dried powders consist ofrespirable particles of diluent, each of which contains at least oneembedded drug nanoparticle.

Freeze-dried powders can be used in DPIs or pMDIs, either alone orcombined with spray-dried nanoparticulate powder. In addition,freeze-dried powders containing drug nanoparticles can be reconstitutedand used in either jet or ultrasonic nebulizers to generate aqueousdispersions that have respirable droplet sizes, where each dropletcontains at least one drug nanoparticle.

One embodiment of the invention is directed to a process and compositionfor propellant-based systems comprising nanoparticulate drug particlesand a surface modifier. Such formulations may be prepared by wet millingthe coarse drug substance and surface modifier in liquid propellant,either at ambient pressure or under high pressure conditions.Alternatively, dry powders containing drug nanoparticles may be preparedby spray-drying or freeze-drying aqueous dispersions of drugnanoparticles and the resultant powders dispersed into suitablepropellants for use in conventional pMDIs. Such nanoparticulate pMDIformulations can be used for either nasal or pulmonary delivery. Forpulmonary administration, such formulations afford increased delivery tothe deep lung regions because of the small (e.g., about 1 to about 2microns MMAD) particle sizes available from these methods. Concentratedaerosol formulations can also be employed in pMDIs.

Another embodiment is directed to dry powders which containnanoparticulate compositions for pulmonary or nasal delivery. Thepowders may consist of respirable aggregates of nanoparticulate drugparticles, or of respirable particles of a diluent which contains atleast one embedded drug nanoparticle. Powders containing nanoparticulatedrug particles can be prepared from aqueous dispersions of nanoparticlesby removing the water via spray-drying or lyophilization (freezedrying). Spray-drying is less time consuming and less expensive thanfreeze-drying, and therefore more cost-effective. However, certaindrugs, such as biologicals benefit from lyophilization rather thanspray-drying in making dry powder formulations.

Conventional micronized drug particles used in dry powder aerosoldelivery having partcticle diameters of from about 2 to about 5 micronsMMAD are often difficult to meter and disperse in small quantitiesbecause of the electrostatic cohesive forces inherent in such powders.These difficulties can lead to loss of drug substance to the deliverydevice as well as incomplete powder dispersion and sub-optimal deliveryto the lung. Many drug compounds, particularly proteins and peptides,are intended for deep lung delivery and systemic absorption. Since theaverage particle sizes of conventionally prepared dry powders areusually in the range of from about 2 to about 5 microns MMAD, thefraction of material which actually reaches the alveolar region may bequite small. Thus, delivery of micronized dry powders to the lung,especially the alveolar region, is generally very inefficient because ofthe properties of the powders themselves.

The dry powder aerosols which contain nanoparticulate drugs can be madesmaller than comparable micronized drug substance and, therefore, areappropriate for efficient delivery to the deep lung. Moreover,aggregates of nanoparticulate drugs are spherical in geometry and havegood flow properties, thereby aiding in dose metering and deposition ofthe administered composition in the lung or nasal cavities.

Dry nanoparticulate compositions can be used in both DPIs and pMDIs. Asused herein, “dry” refers to a composition having less than about 5%water.

In one embodiment, compositions are provided containing nanoparticleswhich have an effective average particle size of less than about 1000nm, more preferably less than about 400 nm, less than about 300 nm, lessthan about 250 nm, or less than about 200 nm, as measured bylight-scattering methods. By “an effective average particle size of lessthan about 1000 nm” it is meant that at least 50% of the drug particleshave a weight average particle size of less than about 1000 nm whenmeasured by light scattering techniques. Preferably, at least 70% of thedrug particles have an average particle size of less than about 1000 nm,more preferably at least 90% of the drug particles have an averageparticle size of less than about 1000 nm, and even more preferably atleast about 95% of the particles have a weight average particle size ofless than about 1000 nm.

For aqueous aerosol formulations, the nanoparticulate agent may bepresent at a concentration of about 5.0 mg/mL up to about 700 mg/mL. Fordry powder aerosol formulations, the nanoparticulate agent may bepresent at a concentration of about 5.0 mg/g up to about 1000 mg/g,depending on the desired drug dosage. Concentrated nanoparticulateaerosols, defined as containing a nanoparticulate drug at aconcentration of about 5.0 mg/mL up to about 700 mg/mL for aqueousaerosol formulations, and about 5.0 mg/g up to about 1000 mg/g for drypowder aerosol formulations, are specifically provided. Suchformulations provide effective delivery to appropriate areas of the lungor nasal cavities in short administration times, ie., less than about3-15 seconds per dose as compared to administration times of up to 4 to20 minutes as found in conventional pulmonary nebulizer therapies.

Nanoparticulate drug compositions for aerosol administration can be madeby, for example, (1) nebulizing a dispersion of a nanoparticulate drug,obtained by either grinding or precipitation; (2) aerosolizing a drypowder of aggregates of nanoparticulate drug and surface modifier (theaerosolized composition may additionally contain a diluent); or (3)aerosolizing a suspension of nanoparticulate drug or drug aggregates ina non-aqueous propellant. The aggregates of nanoparticulate drug andsurface modifier, which may additionally contain a diluent, can be madein a non-pressurized or a pressurized non-aqueous system. Concentratedaerosol formulations may also be made via such methods.

Milling of aqueous drug to obtain nanoparticulate drug may be performedby dispersing drug particles in a liquid dispersion medium and applyingmechanical means in the presence of grinding media to reduce theparticle size of the drug to the desired effective average particlesize. The particles can be reduced in size in the presence of one ormore surface modifiers. Alternatively, the particles can be contactedwith one or more surface modifiers after attrition. Other compounds,such as a diluent, can be added to the drug/surface modifier compositionduring the size reduction process. Dispersions can be manufacturedcontinuously or in a batch mode.

Another method of forming nanoparticle dispersion is bymicroprecipitation. This is a method of preparing stable dispersions ofdrugs in the presence of one or more surface modifiers and one or morecolloid stability enhancing surface active agents free of any tracetoxic solvents or solubilized heavy metal impurities. Such a methodcomprises, for example, (1) dissolving the drug in a suitable solventwith mixing; (2) adding the formulation from step (1) with mixing to asolution comprising at least one surface modifier to form a clearsolution; and (3) precipitating the formulation from step (2) withmixing using an appropriate nonsolvent. The method can be followed byremoval of any formed salt, if present, by dialysis or diafiltration andconcentration of the dispersion by conventional means. The resultantnanoparticulate drug dispersion can be utilized in liquid nebulizers orprocessed to form a dry powder for use in a DPI or pMDI.

In a non-aqueous, non-pressurized milling system, a non-aqueous liquidhaving a vapor pressure of about 1 atm or less at room temperature andin which the drug substance is essentially insoluble may be used as awet milling medium to make a nanoparticulate drug composition. In such aprocess, a slurry of drug and surface modifier may be milled in thenon-aqueous medium to generate nanoparticulate drug particles. Examplesof suitable non-aqueous media include ethanol,trichloromonofluoromethane, (CFC-11), and dichlorotetafluoroethane(CFC-114). An advantage of using CFC-11 is that it can be handled atonly marginally cool room temperatures, whereas CFC-114 requires morecontrolled conditions to avoid evaporation. Upon completion of millingthe liquid medium may be removed and recovered under vacuum or heating,resulting in a dry nanoparticulate composition. The dry composition maythen be filled into a suitable container and charged with a finalpropellant. Exemplary final product propellants, which ideally do notcontain chlorinated hydrocarbons, include HFA-134a (tetrafluoroethane)and HFA-227 (heptafluoropropane). While non-chlorinated propellants maybe preferred for environmental reasons, chlorinated propellants may alsobe used in this aspect of the invention.

In a non-aqueous, pressurized milling system, a non-aqueous liquidmedium having a vapor pressure significantly greater than 1 atm at roomtemperature may be used in the milling process to make nanoparticulatedrug compositions. If the milling medium is a suitable halogenatedhydrocarbon propellant, the resultant dispersion may be filled directlyinto a suitable pMDI container. Alternately, the milling medium can beremoved and recovered under vacuum or heating to yield a drynanoparticulate composition. This composition can then be filled into anappropriate container and charged with a suitable propellant for use ina pMDI.

Spray drying is a process used to obtain a powder containingnanoparticulate drug particles following particle size reduction of thedrug in a liquid medium. In general, spray-drying may be used when theliquid medium has a vapor pressure of less than about 1 atm at roomtemperature. A spray-dryer is a device which allows for liquidevaporation and drug powder collection. A liquid sample, either asolution or suspension, is fed into a spray nozzle. The nozzle generatesdroplets of the sample within a range of about 20 to about 100 μm indiameter which are then transported by a carrier gas into a dryingchamber. The carrier gas temperature is typically from about 80 to about200° C. The droplets are subjected to rapid liquid evaporation, leavingbehind dry particles which are collected in a special reservoir beneatha cyclone apparatus.

If the liquid sample consists of an aqueous dispersion of nanoparticlesand surface modifier, the collected product will consist of sphericalaggregates of the nanoparticulate drug particles. If the liquid sampleconsists of an aqueous dispersion of nanoparticles in which an inertdiluent material was dissolved (such as lactose or mannitol), thecollected product will consist of diluent (e.g., lactose or mannitol)particles which contain embedded nanoparticulate drug particles. Thefinal size of the collected product can be controlled and depends on theconcentration of nanoparticulate drug and/or diluent in the liquidsample, as well as the droplet size produced by the spray-dryer nozzle.Collected products may be used in conventional DPIs for pulmonary ornasal delivery, dispersed in propellants for use in pMDIs, or theparticles may be reconstituted in water for use in nebulizers.

In some instances it may be desirable to add an inert carrier to thespray-dried material to improve the metering properties of the finalproduct. This may especially be the case when the spray dried powder isvery small (less than about 5 μm) or when the intended dose is extremelysmall, whereby dose metering becomes difficult. In general, such carrierparticles (also known as bulking agents) are too large to be deliveredto the lung and simply impact the mouth and throat and are swallowed.Such carriers typically consist of sugars such as lactose, mannitol, ortrehalose. Other inert materials, including polysaccharides andcellulosics, may also be useful as carriers.

Spray-dried powders containing nanoparticulate drug particles may usedin conventional DPIs, dispersed in propellants for use in pMDIs, orreconstituted in a liquid medium for use with nebulizers.

For compounds that are denatured or destabilized by heat, such ascompounds having a low melting point (i.e., about 70 to about 150° C.),or for example, biologics, sublimation is preferred over evaporation toobtain a dry powder nanoparticulate drug composition. This is becausesublimation avoids the high process temperatures associated withspray-drying. In addition, sublimation, also known as freeze-drying orlyophilization, can increase the shelf stability of drug compounds,particularly for biological products. Freeze-dried particles can also bereconstituted and used in nebulizers. Aggregates of freeze-driednanoparticulate drug particles can be blended with either dry powderintermediates or used alone in DPIs and pMDIs for either nasal orpulmonary delivery.

Sublimation involves freezing the product and subjecting the sample tostrong vacuum conditions. This allows for the formed ice to betransformed directly from a solid state to a vapor state. Such a processis highly efficient and, therefore, provides greater yields thanspray-drying. The resultant freeze-dried product contains drug andmodifier(s). The drug is typically present in an aggregated state andcan be used for inhalation alone (either pulmonary or nasal), inconjunction with diluent materials (lactose, mannitol, etc.), in DPIs orpMDIs, or reconstituted for use in a nebulizer.

Liposomal Compositions

In some embodiments, fluoroquinolone antimicrobial agents disclosedherein may be formulated into liposome particles, which can then beaerosolized for inhaled delivery. Lipids which are useful in the presentinvention can be any of a variety of lipids including both neutrallipids and charged lipids. Carrier systems having desirable propertiescan be prepared using appropriate combinations of lipids, targetinggroups and circulation enhancers. Additionally, the compositionsprovided herein can be in the form of liposomes or lipid particles,preferably lipid particles. As used herein, the term “lipid particle”refers to a lipid bilayer carrier which “coats” a nucleic acid and haslittle or no aqueous interior. More particularly, the term is used todescribe a self-assembling lipid bilayer carrier in which a portion ofthe interior layer comprises cationic lipids which form ionic bonds orion-pairs with negative charges on the nucleic acid (e.g., a plasmidphosphodiester backbone). The interior layer can also comprise neutralor fusogenic lipids and, in some embodiments, negatively charged lipids.The outer layer of the particle will typically comprise mixtures oflipids oriented in a tail-to-tail fashion (as in liposomes) with thehydrophobic tails of the interior layer. The polar head groups presenton the lipids of the outer layer will form the external surface of theparticle.

Liposomal bioactive agents can be designed to have a sustainedtherapeutic effect or lower toxicity allowing less frequentadministration and an enhanced therapeutic index. Liposomes are composedof bilayers that entrap the desired pharmaceutical. These can beconfigured as multilamellar vesicles of concentric bilayers with thepharmaceutical trapped within either the lipid of the different layersor the aqueous space between the layers.

By non-limiting example, lipids used in the compositions may besynthetic, semi-synthetic or naturally-occurring lipids, includingphospholipids, tocopherols, steroids, fatty acids, glycoproteins such asalbumin, negatively-charged lipids and cationic lipids. Phosholipidsinclude egg phosphatidylcholine (EPC), egg phosphatidylglycerol (EPG),egg phosphatidylinositol (EPI), egg phosphatidylserine (EPS),phosphatidylethanolamine (EPE), and egg phosphatidic acid (EPA); thesoya counterparts, soy phosphatidylcholine (SPC); SPG, SPS, SPI, SPE,and SPA; the hydrogenated egg and soya counterparts (e.g., HEPC, HSPC),other phospholipids made up of ester linkages of fatty acids in the 2and 3 of glycerol positions containing chains of 12 to 26 carbon atomsand different head groups in the 1 position of glycerol that includecholine, glycerol, inositol, serine, ethanolamine, as well as thecorresponding phosphatidic acids. The chains on these fatty acids can besaturated or unsaturated, and the phospholipid can be made up of fattyacids of different chain lengths and different degrees of unsaturation.In particular, the compositions of the formulations can includedipalmitoylphosphatidylcholine (DPPC), a major constituent ofnaturally-occurring lung surfactant as well asdioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylglycerol(DOPG). Other examples include dimyristoylphosphatidycholine (DMPC) anddimyristoylphosphatidylglycerol (DMPG) dipalmitoylphosphatidcholine(DPPC) and dipalmitoylphosphatidylglycerol (DPPG)distearoylphosphatidylcholine (DSPC) and di stearoylphosphatidylglycerol(DSPG), diol eylphosphatidylethanolamine (DOPE) and mixed phospholipidslike palmitoylstearoylphosphatidylcholine (PSPC) andpalmitoylstearoylphosphatidylglycerol (PSPG), and single acylatedphospholipids like mono-oleoyl-phosphatidylethanolamine (MOPE).

In a preferred embodiment, PEG-modified lipids are incorporated into thecompositions of the present invention as the aggregation-preventingagent. The use of a PEG-modified lipid positions bulky PEG groups on thesurface of the liposome or lipid carrier and prevents binding of DNA tothe outside of the carrier (thereby inhibiting cross-linking andaggregation of the lipid carrier). The use of a PEG-ceramide is oftenpreferred and has the additional advantages of stabilizing membranebilayers and lengthening circulation lifetimes. Additionally,PEG-ceramides can be prepared with different lipid tail lengths tocontrol the lifetime of the PEG-ceramide in the lipid bilayer. In thismanner, “programmable” release can be accomplished which results in thecontrol of lipid carrier fusion. For example, PEG-ceramides havingC₂₀-acyl groups attached to the ceramide moiety will diffuse out of alipid bilayer carrier with a half-life of 22 hours. PEG-ceramides havingC₁₄- and C₈-acyl groups will diffuse out of the same carrier withhalf-lives of 10 minutes and less than 1 minute, respectively. As aresult, selection of lipid tail length provides a composition in whichthe bilayer becomes destabilized (and thus fusogenic) at a known rate.Though less preferred, other PEG-lipids or lipid-polyoxyethyleneconjugates are useful in the present compositions. Examples of suitablePEG-modified lipids include PEG-modified phosphatidylethanolamine andphosphatidic acid, PEG-modified diacylglycerols and dialkylglycerols,PEG-modified dialkylamines and PEG-modified1,2-diacyloxypropan-3-amines. Particularly preferred are PEG-ceramideconjugates (e.g., PEG-Cer-C₈, PEG-Cer-C₁₄ or PEG-Cer-C₂₀) which aredescribed in U.S. Pat. No. 5,820,873, incorporated herein by reference.

The compositions of the present invention can be prepared to provideliposome compositions which are about 50 nm to about 400 nm in diameter.One with skill in the art will understand that the size of thecompositions can be larger or smaller depending upon the volume which isencapsulated. Thus, for larger volumes, the size distribution willtypically be from about 80 nm to about 300 nm.

Surface Modifiers

Fluoroquinolone antimicrobial agents disclosed herein may be prepared ina pharmaceutical composition with suitable surface modifiers which maybe selected from known organic and inorganic pharmaceutical excipients.Such excipients include low molecular weight oligomers, polymers,surfactants and natural products. Preferred surface modifiers includenonionic and ionic surfactants. Two or more surface modifiers can beused in combination.

Representative examples of surface modifiers include cetyl pyridiniumchloride, gelatin, casein, lecithin (phosphatides), dextran, glycerol,gum acacia, cholesterol, tragacanth, stearic acid, benzalkoniumchloride, calcium stearate, glycerol monostearate, cetostearyl alcohol,cetomacrogol emulsifying wax, sorbitan esters, polyoxyethylene alkylethers (e.g., macrogol ethers such as cetomacrogol 1000),polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fattyacid esters (e.g., the commercially available Tweens®, such as e.g.,Tween 20®, and Tween 80®, (ICI Specialty Chemicals)); polyethyleneglycols (e.g., Carbowaxs 3350®, and 1450®, and Carbopol 934®, (UnionCarbide)), dodecyl trimethyl ammonium bromide, polyoxyethylenestearates,colloidal silicon dioxide, phosphates, sodium dodecyl sulfate,carboxymethylcellulose calcium, hydroxypropyl cellulose (HPC, HPC-SL,and HPC-L), hydroxypropyl methylcellulose (HPMC), carboxymethylcellulosesodium, methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose,hydroxypropylmethyl-cellulose phthalate, noncrystalline cellulose,magnesium aluminum silicate, triethanolamine, polyvinyl alcohol (PVA),polyvinylpyrrolidone (PVP), 4-(1,1,3,3-tetaamethylbutyl)-phenol polymerwith ethylene oxide and formaldehyde (also known as tyloxapol,superione, and triton), poloxamers (e.g., Pluronics F68®, and F108®,which are block copolymers of ethylene oxide and propylene oxide);poloxamnines (e.g., Tetronic 908®, also known as Poloxamine 908®, whichis a tetrafunctional block copolymer derived from sequential addition ofpropylene oxide and ethylene oxide to ethylenediamine (BASF WyandotteCorporation, Parsippany, N.J.)); a charged phospholipid such asdimyristoyl phophatidyl glycerol, dioctylsulfosuccinate (DOSS); Tetronic1508®; (T-1508) (BASF Wyandotte Corporation), dialkylesters of sodiumsulfosuccinic acid (e.g., Aerosol OT®, which is a dioctyl ester ofsodium sulfosuccinic acid (American Cyanamid)); Duponol P®, which is asodium lauryl sulfate (DuPont); Tritons X-200®, which is an alkyl arylpolyether sulfonate (Rohm and Haas); Crodestas F-110®, which is amixture of sucrose stearate and sucrose distearate (Croda Inc.);p-isononylphenoxypoly-(glycidol), also known as Olin-Log®, or Surfactant10-G®, (Olin Chemicals, Stamford, Conn.); Crodestas SL-40®, (Croda,Inc.); and SA9OHCO, which is C₁₈H₃₇CH₂(CON(CH₃)—CH₂ (CHOH)₄(CH₂ OH)₂(Eastman Kodak Co.); decanoyl-N-methylglucamide;n-decyl-beta-D-glucopyranoside; n-decyl-beta-D-maltopyranoside;n-dodecyl-beta-D-glucopyranoside; n-dodecyl-beta-D-maltoside;heptanoyl-N-methylglucamide; n-heptyl-beta-D-glucopyranoside;n-heptyl-beta-D-thioglucoside; n-hexyl-beta-D-glucopyranoside;nonanoyl-N-methylglucamide; n-noyl-beta-D-glucopyranoside;octanoyl-N-methylglucarmide; n-octyl-beta-D-glucopyranoside;octyl-beta-D-thioglucopyranoside; and the like. Tyloxapol is aparticularly preferred surface modifier for the pulmonary or intranasaldelivery of steroids, even more so for nebulization therapies.

Examples of surfactants for use in the solutions disclosed hereininclude, but are not limited to, ammonium laureth sulfate, cetamineoxide, cetrimonium chloride, cetyl alcohol, cetyl myristate, cetylpalmitate, cocamide DEA, cocamidopropyl betaine, cocamidopropylamineoxide, cocamide MEA, DEA lauryl sulfate, di-stearyl phthalic acid amide,dicetyl dimethyl ammonium chloride, dipalmitoylethyl hydroxethylmonium,disodium laureth sulfosuccinate, di(hydrogenated) tallow phthalic acid,glyceryl dilaurate, glyceryl distearate, glyceryl oleate, glycerylstearate, isopropyl myristate nf, isopropyl palmitate nf, lauramide DEA,lauramide MEA, lauramide oxide, myristamine oxide, octyl isononanoate,octyl palmitate, octyldodecyl neopentanoate, olealkonium chloride, PEG-2stearate, PEG-32 glyceryl caprylate/caprate, PEG-32 glyceryl stearate,PEG-4 and PEG-150 stearate & distearate, PEG-4 to PEG-150 laurate &dilaurate, PEG-4 to PEG-150 oleate & dioleate, PEG-7 glyceryl cocoate,PEG-8 beeswax, propylene glycol stearate, sodium C14-16 olefinsulfonate, sodium lauryl sulfoacetate, sodium lauryl sulphate, sodiumtrideceth sulfate, stearalkonium chloride, stearamide oxide,TEA-dodecylbenzene sulfonate, TEA lauryl sulfate.

Most of these surface modifiers are known pharmaceutical excipients andare described in detail in the Handbook of Pharmaceutical Excipients,published jointly by the American Pharmaceutical Association and ThePharmaceutical Society of Great Britain (The Pharmaceutical Press,1986), specifically incorporated by reference. The surface modifiers arecommercially available and/or can be prepared by techniques known in theart. The relative amount of drug and surface modifier can vary widelyand the optimal amount of the surface modifier can depend upon, forexample, the particular drug and surface modifier selected, the criticalmicelle concentration of the surface modifier if it forms micelles, thehydrophilic-lipophilic-balance (HLB) of the surface modifier, themelting point of the surface modifier, the water solubility of thesurface modifier and/or drug, the surface tension of water solutions ofthe surface modifier, etc.

In the present invention, the optimal ratio of drug to surface modifieris ˜0.1% to ˜99.9% fluoroquinolone antimicrobial agent, more preferablyabout 10% to about 90%.

Microspheres

Microspheres can be used for pulmonary delivery of fluoroquinolones byfirst adding an appropriate amount of drug compound to be solubilzed inwater. For example, an aqueous levofloxacin solution may be dispersed inmethylene chloride containing a predetermined amount (0.1-1% w/v) ofpoly(DL-lactide-co-glycolide) (PLGA) by probe sonication for 1-3 min onan ice bath. Separately, levofloxacin will be solubilized in methylenechloride containing PLGA (0.1-1% w/v). The resulting water-in-oilprimary emulsion or the polymer/drug solution will be dispersed in anaqueous continuous phase consisting of 1-2% polyvinyl alcohol(previously cooled to 4° C.) by probe sonication for 3-5 min on an icebath. The resulting emulsion will be stirred continuously for 2-4 hoursat room temperature to evaporate methylene chloride. Microparticles thusformed will be separated from the continuous phase by centrifuging at8000-10000 rpm for 5-10 min. Sedimented particles will be washed thricewith distilled water and freeze dried. Freeze-dried levofloxacinmicroparticles will be stored at −20° C.

By non-limiting example, a spray drying approach will be employed toprepare levofloxacin microspheres. An appropriate amount of levofloxacinwill be solubilized in methylene chloride containing PLGA (0.1-1%). Thissolution will be spray dried to obtain the microspheres.

By non-limiting example, levofloxacin microparticles will becharacterized for size distribution (requirement: 90%<5 μm, 95%<10 μm),shape, drug loading efficiency and drug release using appropriatetechniques and methods.

By non-limiting example, this approach may also be used to sequester andimprove the water solubililty of solid, AUC shape-enhancingformulations, such as low-solubility levofloxacin salt forms fornanoparticle-based formulations.

A certain amount of fluoroquinolone can be first dissolved in theminimal quantity of ethanol 96% necessary to maintain thefluoroquinolnoe in solution when diluted with water from 96 to 75%. Thissolution can then be diluted with water to obtain a 75% ethanol solutionand then a certain amount of paracetamol can be added to obtain thefollowing w/w drug/polymer ratios: 1:2, 1:1, 2:1, 3:1, 4:1, 6:1, 9:1,and 19:1. These final solutions are spray-dried under the followingconditions: feed rate, 15 mL/min; inlet temperature, 110° C.; outlettemperature, 85° C.; pressure 4 bar and throughput of drying air, 35m3/hr. Powder is then collected and stored under vacuum in adessiccator.

Solid Lipid Particles

Preparation of fluoroquinolone solid lipid particles may involvedissolving the drug in a lipid melt (phospholipids such as phophatidylcholine and phosphatidyl serine) maintained at least at the meltingtemperature of the lipid, followed by dispersion of the drug-containingmelt in a hot aqueous surfactant solution (typically 1-5% w/v)maintained at least at the melting temeperature of the lipid. The coarsedispersion will be homogenized for 1-10 min using a Microfluidizer® toobtain a nanoemulsion. Cooling the nanoemulsion to a temperature between4-25° C. will re-solidify the lipid, leading to formation of solid lipidnanoparticles. Optimization of formulation parameters (type of lipidmatrix, surfactant concentration and production parameters) will beperformed so as to achieve a prolonged drug delivery. By non-limitingexample, this approach may also be used to sequester and improve thewater solubililty of solid, AUC shape-enhancing formulations, such aslow-solubility levofloxacin salt forms for nanoparticle-basedformulations.

Melt-Extrusion AUC Shape-Enhancing Formulation

Melt-Extrusion AUC shape-enhancing fluoroquinolone formulations may bepreparation by dissolving the drugs in micelles by adding surfactants orpreparing micro-emulsion, forming inclusion complexes with othermolecules such as cyclodextrins, forming nanoparticles of the drugs, orembedding the amorphous drugs in a polymer matrix. Embedding the drughomogeneously in a polymer matrix produces a solid dispersion. Soliddispersions can be prepared in two ways: the solvent method and the hotmelt method. The solvent method uses an organic solvent wherein the drugand appropriate polymer are dissolved and then (spray) dried. The majordrawbacks of this method are the use of organic solvents and the batchmode production process. The hot melt method uses heat in order todisperse or dissolve the drug in an appropriate polymer. Themelt-extrusion process is an optimized version of the hot melt method.The advantage of the melt-extrusion approach is lack of organic solventand continuous production process. As the melt-extrusion is a novelpharmaceutical technique, the literature dealing with it is limited. Thetechnical set-up involves a mixture and extrusion of fluoroquinolone,hydroxypropyl-b-cyclodextrin (HP-b-CD), and hydroxypropylmethylcellulose(HPMC), in order to, by non-limiting example create a AUCshape-enhancing formulation of levofloxacin or other fluoroquinlone.Cyclodextrin is a toroidal-shaped molecule with hydroxyl groups on theouter surface and a cavity in the center. Cyclodextrin sequesters thedrug by forming an inclusion complex. The complex formation betweencyclodextrins and drugs has been investigated extensively. It is knownthat water-soluble polymer interacts with cyclodextrin and drug in thecourse of complex formation to form a stabilized complex of drug andcyclodextrin co-complexed with the polymer. This complex is more stablethan the classic cyclodextrin-drug complex. As one example, HPMC iswater soluble; hence using this polymer with HP-b-CD in the melt isexpected to create an aqueous soluble AUC shape-enhancing formulation.By non-limiting example, this approach may also be used to sequester andimprove the water solubililty of solid, AUC shape-enhancingformulations, such as low-solubility levofloxacin salt forms fornanoparticle-based formulations.

Co-Precipitates

Co-precipitate fluoroquinolone formulations may be prepared by formationof co-precipitates with pharmacologically inert, polymeric materials. Ithas been demonstrated that the formation of molecular solid dispersionsor co-precipitates to create an AUC shape-enhancing formulations withvarious water-soluble polymers can significantly slow their in vitrodissolution rates and/or in vivo absorption. In preparing powderedproducts, grinding is generally used for reducing particle size, sincethe dissolution rate is strongly affected by particle size. Moreover, astrong force (such as grinding) may increase the surface energy andcause distortion of the crystal lattice as well as reducing particlesize. Co-grinding drug with hydroxypropylmethylcellulose,b-cyclodextrin, chitin and chitosan, crystalline cellulose, andgelatine, may enhance the dissolution properties such that AUCshape-enhancement is obtained for otherwise readily bioavailablefluoroquinolones. By non-limiting example, this approach may also beused to sequester and improve the water solubility of solid, AUCshape-enhancing formulations, such as low-solubility levofloxacin saltforms for nanoparticle-based formulations.

Dispersion-Enhancing Peptides

Compositions may include one or more di- or tripeptides containing twoor more leucine residues. By further non-limiting example, U.S. Pat. No.6,835,372 disclosing dispersion-enhancing peptides, is herebyincorporated by reference in its entirety. This patent describes thediscovery that di-leucyl-containing dipeptides (e.g., dileucine) andtripeptides are superior in their ability to increase the dispersibilityof powdered composition.

In another embodiment, highly dispersible particles including an aminoacid are administered. Hydrophobic amino acids are preferred. Suitableamino acids include naturally occurring and non-naturally occurringhydrophobic amino acids. Some naturally occurring hydrophobic aminoacids, including but not limited to, non-naturally occurring amino acidsinclude, for example, beta-amino acids. Both D, L and racemicconfigurations of hydrophobic amino acids can be employed. Suitablehydrophobic amino acids can also include amino acid analogs. As usedherein, an amino acid analog includes the D or L configuration of anamino acid having the following formula: —NH—CHR—CO—, wherein R is analiphatic group, a substituted aliphatic group, a benzyl group, asubstituted benzyl group, an aromatic group or a substituted aromaticgroup and wherein R does not correspond to the side chain of anaturally-occurring amino acid. As used herein, aliphatic groups includestraight chained, branched or cyclic C1-C8 hydrocarbons which arecompletely saturated, which contain one or two heteroatoms such asnitrogen, oxygen or sulfur and/or which contain one or more units ofdesaturation. Aromatic groups include carbocyclic aromatic groups suchas phenyl and naphthyl and heterocyclic aromatic groups such asimidazolyl, indolyl, thienyl, furanyl, pyridyl, pyranyl, oxazolyl,benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl and acridintyl.

Suitable substituents on an aliphatic, aromatic or benzyl group include—OH, halogen (—Br,—Cl,—I and —F), —O(aliphatic, substituted aliphatic,benzyl, substituted benzyl, aryl or substituted aryl group),—CN, —NO₂,—COOH, —NH₂, —NH(aliphatic group, substituted aliphatic, benzyl,substituted benzyl, aryl or substituted aryl group), —N(aliphatic group,substituted aliphatic, benzyl, substituted benzyl, aryl or substitutedaryl group)₂, —COO(aliphatic group, substituted aliphatic, benzyl,substituted benzyl, aryl or substituted aryl group), —CONH₂,—CONH(aliphatic, substituted aliphatic group, benzyl, substitutedbenzyl, aryl or substituted aryl group)), —SH, —S(aliphatic, substitutedaliphatic, benzyl, substituted benzyl, aromatic or substituted aromaticgroup) and —NH—C(.dbd.NH)—NH₂. A substituted benzylic or aromatic groupcan also have an aliphatic or substituted aliphatic group as asubstituent. A substituted aliphatic group can also have a benzyl,substituted benzyl, aryl or substituted aryl group as a substituent. Asubstituted aliphatic, substituted aromatic or substituted benzyl groupcan have one or more substituents. Modifying an amino acid substituentcan increase, for example, the lypophilicity or hydrophobicity ofnatural amino acids which are hydrophilic.

A number of the suitable amino acids, amino acids analogs and saltsthereof can be obtained commercially. Others can be synthesized bymethods known in the art.

Hydrophobicity is generally defined with respect to the partition of anamino acid between a nonpolar solvent and water. Hydrophobic amino acidsare those acids which show a preference for the nonpolar solvent.Relative hydrophobicity of amino acids can be expressed on ahydrophobicity scale on which glycine has the value 0.5. On such ascale, amino acids which have a preference for water have values below0.5 and those that have a preference for nonpolar solvents have a valueabove 0.5. As used herein, the term hydrophobic amino acid refers to anamino acid that, on the hydrophobicity scale, has a value greater orequal to 0.5, in other words, has a tendency to partition in thenonpolar acid which is at least equal to that of glycine.

Examples of amino acids which can be employed include, but are notlimited to: glycine, proline, alanine, cysteine, methionine, valine,leucine, tyosine, isoleucine, phenylalanine, tryptophan. Preferredhydrophobic amino acids include leucine, isoleucine, alanine, valine,phenylalanine and glycine. Combinations of hydrophobic amino acids canalso be employed. Furthermore, combinations of hydrophobic andhydrophilic (preferentially partitioning in water) amino acids, wherethe overall combination is hydrophobic, can also be employed.

The amino acid can be present in the particles of the invention in anamount of at least 10 weight %. Preferably, the amino acid can bepresent in the particles in an amount ranging from about 20 to about 80weight %. The salt of a hydrophobic amino acid can be present in theparticles of the invention in an amount of at least 10 weight percent.Preferably, the amino acid salt is present in the particles in an amountranging from about 20 to about 80 weight %. In preferred embodiments theparticles have a tap density of less than about 0.4 g/cm.sup.3.

Methods of forming and delivering particles which include an amino acidare described in U.S. Pat. No. 6,586,008, entitled Use of Simple AminoAcids to Form Porous Particles During Spray Drying, the teachings ofwhich are incorporated herein by reference in their entirety.

Proteins/Amino Acids

Protein excipients may include albumins such as human serum albumin(HSA), recombinant human albumin (rHA), gelatin, casein, hemoglobin, andthe like. Suitable amino acids (outside of the dileucyl-peptides of theinvention), which may also function in a buffering capacity, includealanine, glycine, arginine, betaine, histidine, glutamic acid, asparticacid, cysteine, lysine, leucine, isoleucine, valine, methionine,phenylalanine, aspartame, tyrosine, tryptophan, and the like. Preferredare amino acids and polypeptides that function as dispersing agents.Amino acids falling into this category include hydrophobic amino acidssuch as leucine, valine, isoleucine, tryptophan, alanine, methionine,phenylalanine, tyrosine, histidine, and proline.Dispersibility-enhancing peptide excipients include dimers, trimers,tetramers, and pentamers comprising one or more hydrophobic amino acidcomponents such as those described above.

Carbohydrates

By non-limiting example, carbohydrate excipients may includemonosaccharides such as fructose, maltose, galactose, glucose,D-mannose, sorbose, and the like; disaccharides, such as lactose,sucrose, trehalose, cellobiose, and the like; polysaccharides, such asraffinose, melezitose, maltodextrins, dextrans, starches, and the like;and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitolsorbitol (glucitol), pyranosyl sorbitol, myoinositol, isomalt, trehaloseand the like.

Polymers

By non-limiting example, compositions may also include polymericexcipients/additives, e.g., polyvinylpyrrolidones, derivatizedcelluloses such as hydroxymethylcellulose, hydroxyethyl cellulose, andhydroxypropylmethylcellulose, Ficolls (a polymeric sugar),hydroxyethylstarch, dextrates (by non-limiting example cyclodextrins mayinclude, 2-hydroxypropyl-beta-cyclodextrin,2-hydroxypropyl-gamma-cyclodextrin, randomly methylatedbeta-cyclodextrin, dimethyl-alpha-cyclodextrin,dimethyl-beta-cyclodextrin, maltosyl-alpha-cyclodextrin,glucosyl-1-alpha-cyclodextrin, glucosyl-2-alpha-cyclodextrin,alpha-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin, andsulfobutylether-b eta-cyclodextrin), polyethylene glycols, and pectinmay also be used.

Highly dispersible particles administered comprise a bioactive agent anda biocompatible, and preferably biodegradable polymer, copolymer, orblend. The polymers may be tailored to optimize differentcharacteristics of the particle including: i) interactions between theagent to be delivered and the polymer to provide stabilization of theagent and retention of activity upon delivery; ii) rate of polymerdegradation and, thereby, rate of drug release profiles; iii) surfacecharacteristics and targeting capabilities via chemical modification;and iv) particle porosity.

Surface eroding polymers such as polyanhydrides may be used to form theparticles. For example, polyanhydrides such aspoly[(p-carboxyphenoxy)hexane anhydride] (PCPH) may be used.Biodegradable polyanhydrides are described in U.S. Pat. No. 4,857,311.Bulk eroding polymers such as those based on polyesters includingpoly(hydroxy acids) also can be used. For example, polyglycolic acid(PGA), polylactic acid (PLA), or copolymers thereof may be used to formthe particles. The polyester may also have a charged or functionalizablegroup, such as an amino acid. In a preferred embodiment, particles withcontrolled release properties can be formed of poly(D,L-lactic acid)and/or poly(DL-lactic-co-glycolic acid) (“PLGA”) which incorporate asurfactant such as dipalmitoyl phosphatidylcholine (DPPC).

Other polymers include polyamides, polycarbonates, polyalkylenes such aspolyethylene, polypropylene, poly(ethylene glycol), poly(ethyleneoxide), poly(ethylene terephthalate), poly vinyl compounds such aspolyvinyl alcohols, polyvinyl ethers, and polyvinyl esters, polymers ofacrylic and methacrylic acids, celluloses and other polysaccharides, andpeptides or proteins, or copolymers or blends thereof. Polymers may beselected with or modified to have the appropriate stability anddegradation rates in vivo for different controlled drug deliveryapplications.

Highly dispersible particles can be formed from functionalized polyestergraft copolymers, as described in Hrkach et al., Macromolecules, 28:4736-4739 (1995); and Hrkach et al., “Poly(L-Lactic acid-co-amino acid)Graft Copolymers: A Class of Functional Biodegradable Biomaterials” inHydrogels and Biodegradable Polymers for Bioapplications, ACS SymposiumSeries No. 627, Raphael M, Ottenbrite et al., Eds., American ChemicalSociety, Chapter 8, pp. 93-101, 1996.

In a preferred embodiment of the invention, highly dispersible particlesincluding a bioactive agent and a phospholipid are administered.Examples of suitable phospholipids include, among others,phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols,phosphatidylserines, phosphatidylinositols and combinations thereof.Specific examples of phospholipids include but are not limited tophosphatidylcholines dipalmitoyl phosphatidylcholine (DPPC), dipalmitoylphosphatidylethanolamine (DPPE), distearoyl phosphatidyicholine (DSPC),dipalmitoyl phosphatidyl glycerol (DPPG) or any combination thereof.Other phospholipids are known to those skilled in the art. In apreferred embodiment, the phospholipids are endogenous to the lung.

The phospholipid, can be present in the particles in an amount rangingfrom about 0 to about 90 weight %. More commonly it can be present inthe particles in an amount ranging from about 10 to about 60 weight %.

In another embodiment of the invention, the phospholipids orcombinations thereof are selected to impart controlled releaseproperties to the highly dispersible particles. The phase transitiontemperature of a specific phospholipid can be below, around or above thephysiological body temperature of a patient. Preferred phase transitiontemperatures range from 30 degrees C. to 50 degrees C. (e.g., within+/−10 degrees of the normal body temperature of patient). By selectingphospholipids or combinations of phospholipids according to their phasetransition temperature, the particles can be tailored to have controlledrelease properties. For example, by administering particles whichinclude a phospholipid or combination of phospholipids which have aphase transition temperature higher than the patient's body temperature,the release of dopamine precursor, agonist or any combination ofprecursors and/or agonists can be slowed down. On the other hand, rapidrelease can be obtained by including in the particles phospholipidshaving lower transition temperatures.

Taste Masking, Flavor, Other

By non-limiting example, compositions may further include flavoringagents, taste-masking agents, inorganic salts (e.g., sodium chloride),antimicrobial agents (e.g., benzalkonium chloride), sweeteners,antioxidants, antistatic agents, surfactants (e.g., polysorbates such as“TWEEN 20” and “TWEEN 80”), sorbitan esters, saccharin, cyclodextrins,lipids (e.g., phospholipids such as lecithin and otherphosphatidylcholines, phosphatidylethanolamines), fatty acids and fattyesters, steroids (e.g., cholesterol), and chelating agents (e.g., EDTA,zinc and other such suitable cations). Other pharmaceutical excipientsand/or additives suitable for use in the compositions according to theinvention are listed in “Remington: The Science & Practice of Pharmacy”,19th ed., Williams & Williams, (1995), and in the “Physician's DeskReference”, 52nd ed., Medical Economics, Montvale, N.J. (1998).

By non-limiting example, classes of taste-masking agents forfluoroquinolone formulation include the addition of flavorings,sweeteners, and other various coating strategies. By non-limitingexamples these may be chosen from sugars such as sucrose, dextrose, andlactose), carboxylic acids, salts such as magnesium and calcium(non-specific or chelation-based fluoroquinolone taste masking),menthol, amino acids or amino acid derivatives such as arginine, lysine,and monosodioum glutamate, and synthetic flavor oils and flavoringaeromatics and/or natural oils, extracts from plants, leaves, flowers,fruits, etc. and combinations thereof. These may include cinnamon oils,oil of wintergreen, peppermint oils, clover oil, bay oil, anise oil,eucalyptus, vanilla, citrus oil such as lemon oil, orange oil, grape andgrapefruit oil, fruit essences including apple, peach, pear, strawberry,raspberry, cherry, plum, pineapple, apricot, etc. Additional sweetenersinclude sucrose, dextrose, aspartame (Nutrasweet®), acesulfame-K,sucrolose and saccharin, organic acids (by non-limiting example citricacid and aspartic acid). Such flavors may be present at about 0.05 toabout 4 percent. Another approach to improve or mask the taste ofunpleasant inhaled drugs is to decrease the drugs solubility, e.g. drugsmust dissolve to interact with taste receptors. Hence, to deliver solidforms of the drug may avoid the taste response and acquire the desiredimproved taste affect. Non-limiting methods to decrease fluoroquinolonesolubility are described in this document, e.g. salt forms oflevofloxacin or gemifloxacin with xinafoic acid, oleic acid, stearicacid and pamoic acid. Additional co-precipitating agents includedihydropyridines and a polymer such as polyvinyl pyrrolidone. Moreover,taste-masking may be accomplished by creation of lipopilic vesicles.Additional coating or capping agents include dextrates (by non-limitingexample cyclodextrins may include, 2-hydroxypropyl-beta-cyclodextrin,2-hydroxypropyl-gamma-cyclodextrin, randomly methylatedbeta-cyclodextrin, dimethyl-alpha-cyclodextrin,dimethyl-beta-cyclodextrin,malto syl-alp ha-cyclo dextrin,glucosyl-1-alpha-cyclodextrin, glucosyl-2-alpha-cyclodextrin,alpha-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin, andsulfobutylether-beta-cyclodextrin), modified celluloses such as ethylcellulose, methyl cellulose, hydroxypropyl cellulose, hydroxyl propylmethyl cellulose, polyalkylene glycols, polyalkylene oxides, sugars andsugar alcohols, waxes, shellacs, acrylics and mixtures thereof. Bynon-limiting example, other methods to deliver non-dissolved forms offluoroquinolones are to administer the drug alone or in simple,non-solubilty affecting formulation as a crystalline micronized, drypowder, spray-dried, and nanosuspension formulation. However, analternative method is to include taste-modifying agents. These includetaste-masking substance that is mixd with, coated onto or otherwisecombined with the fluoroquinolone active medicament. However, thisaddition may also serve to improve the taste of another chosen drugproduct addition, e.g. a mucolytic agent. Non-limiting examples of suchsubstances include acid phospholipids, lysophospholipid, tocopherolpolyethyleneglycol succinate, and embonic acid (pamoate). Many of theseagents can be used alone or in combination with fluoroquinolones foraerosol administration.

Mucolytic Agents

Methods to produce formulations that combine agents to reduce sputumviscosity during aerosol treatment with a fluoroquinolone include thefollowing. These agents can be prepared in fixed combination or beadministered in succession with aerosol fluoroquinolone therapy.

The most commonly prescribed agent is N-acetylcysteine (NAC), whichdepolymerizes mucus in vitro by breaking disulphide bridges betweenmacromolecules. It is assumed that such reduction of sputum tenacityfacilitates its removal from the respiratory tract. In addition, NAC mayact as an oxygen radical scavenger. NAC can be taken either orally or byinhalation. Differences between these two methods of administration havenot been formally studied. After oral administration, NAC is reduced tocysteine, a precursor of the antioxidant glutathione, in the liver andintestine. The antioxidant properties could be useful in preventingdecline of lung function in cystic fibrosis (CF). Nebulized NAC iscommonly prescribed to patients with CF, in particular in continentalEurope, in order to improve expectoration of sputum by reducing itstenacity. The ultimate goal of this is to slow down the decline of lungfunction in CF.

L-lysine-N-acetylcysteinate (ACC) or Nacystelyn (NAL) is a novelmucoactive agent possessing mucolytic, antioxidant, andanti-inflammatory properties. Chemically, it is a salt of ACC. This drugappears to present an activity superior to its parent molecule ACCbecause of a synergistic mucolytic activity of L-lysine and ACC.Furthermore, it's almost neutral pH (6.2) allows its administration inthe lungs with a very low incidence of bronchospasm, which is not thecase for the acidic ACC (pH 2.2). NALis difficult to formulate in aninhaled form because the required lung dose is very high (approximately2 mg) and the micronized drug is sticky and cohesive and it is thusproblematic to produce a redispersable formulation. NAL was firstdeveloped as a chlorofluorocarbon (CFC) containing metered-dose inhaler(MDI) because this form was the easiest and the fastest to develop tobegin the preclinical and the first clinical studies. NAL MDI delivered2 mg per puff, from which approximately 10% was able to reach the lungsin healthy volunteers. One major inconvenience of this formulation waspatient compliance because as many as 12 puffs were necessary to obtainthe required dose. Furthermore, the progressive removal of CFC gasesfrom medicinal products combined with the problems of coordination metin a large proportion of the patient population (12) have led to thedevelopment of a new galenical form of NAL. A dry powder inhaler (DPI)formulation was chosen to resolve the problems of compliance with MDIsand to combine it with an optimal, reproducible, and comfortable way toadminister the drug to the widest possible patient population, includingyoung children.

The DPI formulation of NAL involved the use of a nonconventional lactose(usually reserved for direct compression of tablets), namely, aroller-dried (RD) anhydrous (3-lactose. When tested in vitro with amonodose DPI device, this powder formulation produces a fine particlefraction (FPF) of at least 30% of the nominal dose, namely three timeshigher than that with MDIs. This approach may be used in combinationwith a fluoroquinolone antibiotic for either co-administration or fixedcombination administration antibiotic therapy.

In addition to mucolytic activity, excessive neutrophil elastaseactivity within airways of cystic fibrosis (CF) patients results inprogressive lung damage. Disruption of disulfide bonds on elastase byreducing agents may modify its enzymatic activity. Three naturallyoccurring dithiol reducing systems were examined for their effects onelastase activity: 1) Escherichia coli thioredoxin (Trx) system, 2)recombinant human thioredoxin (rhTrx) system, and 3) dihydrolipoic acid(DHLA). The Trx systems consisted of Trx, Trx reductase, and NADPH. Asshown by spectrophotometric assay of elastase activity, the two Trxsystems and DHLA inhibited purified human neutrophil elastase as well asthe elastolytic activity present in the soluble phase (sol) of CFsputum. Removal of any of the three Trx system constituents preventedinhibition. Compared with the monothiols N-acetylcysteine and reducedglutathione, the dithiols displayed greater elastase inhibition. Tostreamline Trx as an investigational tool, a stable reduced form ofrhTrx was synthesized and used as a single component. Reduced rhTrxinhibited purified elastase and CF sputum sol elastase without NADPH orTrx reductase. Because Trx and DHLA have mucolytic effects, weinvestigated changes in elastase activity after mucolytic treatment.Unprocessed CF sputum was directly treated with reduced rhTrx, the Trxsystem, DHLA, or DNase. The Trx system and DHLA did not increaseelastase activity, whereas reduced rhTrx treatment increased solelastase activity by 60%. By contrast, the elastase activity after DNasetreatment increased by 190%. The ability of Trx and DHLA to limitelastase activity combined with their mucolytic effects makes thesecompounds potential therapies for CF.

In addition, bundles of F-actin and DNA present in the sputum of cysticfibrosis (CF) patients but absent from normal airway fluid contribute tothe altered viscoelastic properties of sputum that inhibit clearance ofinfected airway fluid and exacerbate the pathology of CF. One approachto alter these adverse properties is to remove these filamentousaggregates using DNase to enzymatically depolymerize DNA to constituentmonomers and gelsolin to sever F-actin to small fragments. The highdensities of negative surface charge on DNA and F-actin suggest that thebundles of these filaments, which alone exhibit a strong electrostaticrepulsion, may be stabilized by multivalent cations such as histones,antimicrobial peptides, and other positively charged molecules prevalentin airway fluid. Furthermore, As a matter-a-fact, it has been observedthat bundles of DNA or F-actin formed after addition of histone H1 orlysozyme are efficiently dissolved by soluble multivalent anions such aspolymeric aspartate or glutamate. Addition of poly-aspartate orpoly-glutamate also disperses DNA and actin-containing bundles in CFsputum and lowers the elastic moduli of these samples to levelscomparable to those obtained after treatment with DNase I or gelsolin.Addition of poly-aspartic acid also increased DNase activity when addedto samples containing DNA bundles formed with histone H1. When added toCF sputum, poly-aspartic acid significantly reduced the growth ofbacteria, suggesting activation of endogenous antibacterial factors.These findings suggest that soluble multivalent anions have potentialalone or in combination with other mucolytic agents to selectivelydissociate the large bundles of charged biopolymers that form in CFsputum.

Hence, NAC, unfractionated heparin, reduced glutathione, dithiols, Trx,DHLA, other monothiols, DNAse, dornase alfa, hypertonic formulations(e.g. osmolalities greater than about 350 mOsmol/kg), multivalent anionssuch as polymeric aspartate or glutamate, glycosidases and otherexamples listed above can be combined with fluoroquinolone antibioticsand other mucolytic agents for aerosol administration to improveantibacterial activity through better distribution from reduced sputumviscosity, and improved clinical outcome through improved pulmonaryfunction (from improved sputum mobility and mucociliary clearance) anddecreased lung tissue damage from the immune inflammatory response.

EXAMPLES

The following examples serve to more fully describe the manner of usingthe above-described invention, as well as to set forth the best modescontemplated for carrying out various aspects of the invention. It isunderstood that these examples in no way serve to limit the true scopeof this invention, but rather are presented for illustrative purposes.All references cited herein are incorporated by reference in theirentirety.

Example 1 High Local Concentration with Short Duration AerosolFluoroquinolone Exposure

Aerosol administration of fluoroquinolones such as levofloxacin produceshigh concentrations in the epithelial lining fluid (ELF) of rats andhumans. However, this dose has been observed to rapidly declinefollowing administration.

In order to determine if short duration, high concentrations oflevofloxacin could be effective in treatment of P. aeruginosa (PA),studies were conducted to measure their bactericidal activity on variousstrains of this organism which were grown at different conditions. Thosewere chosen based on what is known about conditions and growth of PA ina cystic fibrosis (CF) lung. Four isogenic strains of P. aeruginosa wereused for these experiments (Table 2).

TABLE 2 Strains of PA Used in Time-Kill Experiments Levofloxacin MICStrain Genotype (ug/ml) PAM1020 Wt 0.25 PAM1032 nalB 1

PAM1020 is the parent wild-type strain, PAM1032 contains nalB mutationwhich results in increased levofloxacin resistance due to overexpressionof the MexAB-OprM efflux pump which can extrude levofloxacin out ofcells.

Experiment 1. Activity of Levofloxacin Against Exponentially GrownCells.

Methods

Inoculum Preparation

Strains were grown aerobically overnight in Mueller-Hinton Broth (MHB)at 35° C. Next, cultures were diluted 1:1000 into 100 ml of fresh MHBand grown to OD₆₀₀˜0.3 to reach CFU/ml ˜10⁸. 10 ml of this culture wasmoved to 50 ml flasks, each containing 10 ml of pre-warmed MHB brothwith appropriate concentrations of levofloxacin (2× as compared to theexposure concentrations).

Exposure

All strains were treated for 10 min., 20 min., 40 min., 80 min. and 160minutes. The following concentrations of levofloxacin (ug/ml) were usedfor the exposure of PAM1020 and PAM1032: 16, 32, 64, 128 and 256. Allstrains were treated at each concentration for 10 min., 20 min., 40min., 80 min. and 160 minutes.

Determination of Viable Cell Numbers

At appropriate times, 1 ml of each exposure culture was centrifuged for2 minutes, the pellet was washed twice with 1 ml of drug-free MHB, andre-suspended in 1 ml of MHB. The viable cell numbers were enumerated byplating serially diluted samples (in duplicates) on MHB plates by thedrop (10 ul) plating method. The limit of detection was 100 CFU/ml.Killing is reported as the log reduction calculated relative to cellcount at the time of initiation of antibiotic exposure. Relativeantibiotic concentrations (relative to MIC of the corresponding strains)are used. Cell numbers at initiation of antibiotic exposure are shown inTable 3.

TABLE 3 Bacterial Numbers at Time of Initial Bacterial Exposure StrainCFU/ml PAM1020 4.03E+07 PAM1032 5.60E+07

Results

For the most susceptible strain, PAM1020, maximum killing (5.5 logdecrease in viable cell counts) was achieved after incubation for 10minutes with the concentration of levofloxacin corresponding to 256-foldMIC (64 ug/ml tested). 5-logs of killing were achieved already with thelowest concentration tested (16 ug/ml or 64-fold MIC) (FIG. 4A). For thestrain PAM1032, as long as the concentration above 128-fold the MIC (128ug/ml) was reached, 10 minute of exposure was sufficient to result inmaximum killing (more than 5 logs). At short duration exposures (10 or20 minutes), less killing was observed at concentrations below 128-foldof MICs. At longer exposure times, concentration corresponding to16-fold MICs and above resulted in similar maximum killing (FIG. 4B).These results inducate that logarithmic cells of P. aeruginosa areefficiently killed after short duration exposures to high concentrationsof levofloxacin.

Experiment 2. Activity of Levofloxacin Against Stationary Phase Cells

Methods

Inoculum Preparation

Strains were grown aerobically overnight in Mueller-Hinton Broth (MHB)at 35° C. (350 ml total). The spent medium was obtained aftercentrifugation of overnight cultures and filtering the supernatant.Cultures were diluted to OD=0.3 into spent medium. The same medium wasalso used to prepare antibiotic concentrations (the same as inExperiment 1).

Exposure

Antibiotic concentrations, time of exposure as determination of viablecell counts were the same as in Experiment 1.

Results

Cell numbers at initiation of antibiotic exposure are shown in Table 4.

TABLE 4 Bacterial Numbers at Time of Initial Bacterial Exposure StrainCFU/ml PAM1020  8.0E+08 PAM1032 8.50E+08

For stationary phase cells of PAM1020, maximum killing was observed atthe lowest concentration corresponding to 64-fold above MIC (16 ug/ml)and the shortest duration of exposure, 10 minutes (FIG. 5A). However,PAM1032 demonstrated clear dose-dependent killing with the maximumkilling (4 logs) at concentrations 64 the MIC at a short exposure time.Extending the exposure times did not result in larger extent of killing.However, lower concentrations of drug were required to achieve the samekilling at longer exposure times (FIG. 5B).

Next, we have compared the re-growth of PAM1020 and PAM1032 after either10 minutes or 160 minutes of treatment with various concentrations oflevofloxacin. After the corresponding treatments, cells were washedtwice with antibiotic-free medium. 150 μl of cells was placed into96-well plate and the growth was continuously monitored at A₆₆₀ usingSpectraMax (Molecular Devices). The results are shown in FIGS. 6A-6D.

The results demonstrate that the re-growth of both strains was observedat approximately the same time whether cells were treated with highconcentrations of levofloxacin for 10 minutes or 160 minutes. Theseresults further support the efficiency of short duration treatment withhigh concentrations of levofloxacin.

Experiment 3. Activity of Levofloxacin Against Cells Grown UnderOxygen-Limiting Conditions.

Methods

Inoculum Preparation

Overnight cultures were grown aerobically overnight in Mueller-HintonBroth and next diluted 1:10000 in MHB which filled growth flasks to thevery top. Cultures were grown without shaking to OD˜0.3 at 37° C. Underthese conditions an average of ˜20 hours was required to reach an OD=0.3as compared to ˜5 hours under aerated conditions (50 ml of medium in 250ml flasks, vigorous shaking). Upon analysis, it appeared that an OD=0.3corresponded to a late-logarithmic phase of growth. Other than decreasedaeration, antibiotic concentration, time of exposure, and determinationof viable cell counts were the same as in Experiments 1 and 2.

Results

Cell numbers at initiation of antibiotic exposure are shown in Table 5.

TABLE 5 Bacterial Numbers at Time of Initial Bacterial Exposure StrainCFU/ml PAM1020 7.5E+07 PAM1032 8.5E+07

In the case of PAM1020 near maximum killing (4 logs vs 4.5 logs observedunder normal aeration) was achieved after exposure with the lowestconcentration of levofloxacin for the shortest duration of time (10minutes) (FIG. 7A). In the case of PAM1032 dose-dependent killing wasobserved for 10 minutes or 20 minutes of exposure with the highestkilling observed at concentrations corresponding to 128 to 256-fold theMIC. Slightly stronger (less than 1 log difference) killing was observedfor longer exposure intervals (FIG. 7B). These data indicate that underconditions of oxygen limitation cells which are at the late logarithmicphase of growth are efficiently killed after short duration of exposurewith high concentrations of levofloxacin.

Experiment 4. Activity of Levofloxacin Against PAM1032 CF Sputum.

Methods

Cells of strain PAM1032 (MIC=1 ug/ml) were grown to OD=1(late-exponential/early stationary phase of growth) in MHB and nextconcentrated 10-fold in 10-fold concentrated MHB. 10 ul of cells werethen added to 90 ul of sputum or water in 96-well round bottom plates,restoring MHB to its original concentration. Quantitation plates werepre-warmed for 5 minutes at 37° C. and different concentrations oflevofloxacin (512 ug/ml, 128 ug/ml, 32 ug/ml, 8 ug/ml, 2 ug/ml, and 0.5ug/ml) were added. At appropriate times, 10 ul of each treatment culturewas diluted 100-fold in MHB to minimize the carryover of levofloxacin.Viable cell numbers were enumerated by plating serially diluted sampleson MHB plates by the drop (10 ul) plating method. The limit of detectionwas 10⁴ CFU/ml. Killing is reported as the percentage of the startinginoculum survived after the levofloxacin treatment. Results are shown inFIGS. 8A and 8B.

Results

The results indicate that while sputum slightly affected the degree ofkilling by levofloxacin, rapid and extensive (up to five orders ofmagnitude) killing by levofloxacin in sputum was still observed aftershort duration of treatment at high concentrations of antibiotic.

Experiment 4. Activity of Levofloxacin Against Colony Biofilms ofPAM1032.

Methods

Biofilm Preparation

Colony biofilms were grown on polycarbonate membrane filters (diameter,25 mm; Poretics, Livermore, Calif.) resting on MHB plates. Overnightculture of PAM1032 was diluted to OD=0.3, and then diluted 1:100 infresh MHB. 5 ul of this culture was spotted on the membrane filter.Bacteria were incubated at 37° C. for 48 hours (mature biofilms).

Exposure

After growth filters were placed into tubes containing 3 ml saline orsaline and levofloxacin at 128 ug/ml and 1024 ug/ml. Each tube wastreated for 10 minutes and 80 minutes. At about 5 min. before incubationtime elapsed, tubes were vigorously vortexed (A) or sonicated andvortexed (B) to detach cells. 1 ml of each exposure culture wascentrifuged for 2 minutes, the pellet was washed twice with 1 ml ofdrug-free MHB, and re-suspended in 1 ml of MHB. The viable cell numberswere enumerated by plating serially diluted samples (in duplicates) onMHB plates by the drop (10 ul) plating method. Results are shown in FIG.9.

Results

The data demonstrate that maximum killing (˜2 logs) is obtained after 10min with the lowest concentration of levofloxacin tested (128-fold MIC).No additional killing was observed at the higher levofloxacinconcentration. These data indicate that colony biofilms are moreresistant to killing as compared to logarithmic or stationary phasecells. However, the maximum observed bactericidal activity againstbiofilms (99% under these conditions) was achieved after 10 minutes oflevofloxacin exposure.

Experiment 5. Simulated Short-Term, Rapid Aerosol Administration,Delivering High Concentration Drug Exposure in in vitro PharmacodynamicModel.

In vitro pharmacodynamic models of infection allow for exposure of agrowing bacterial inoculum to changing concentrations of drug as wouldoccur in vivo. The strength of this approach is that the serumconcentration vs. time profile of a drug in man can be simulated in thelaboratory in vitro to determine the optimal exposure profile (i.e.,dose and dosing interval) for a drug and target pathogen.

The following report describes experiments designed to determine theCmax and AUC that will provide maximum bactericidal effects after anaerosol dose of a fluoroquinolone.

Material and Methods

In Vitro Pharmacodynamic Model of Infection

The in vitro pharmacodynamic model consists of a central (analogous“serum” compartment) and peripheral (“extravascular”) compartment. Theperipheral compartments consist of artificial capillary units (Unisyn,Hopkinton, Mass.) arranged in series with the central compartment. Eachcapillary unit has a bundle of small semi-permeable fibers with amolecular size retention of ca. 10,000 MW to allow passage of nutrientsbut not bacteria. The entire system is set up in a dry heat incubatoradjusted to 37° C.

Both the central and peripheral compartments were filled withMueller-Hinton broth. Each peripheral compartment (capillary unit andtubing) contained ca. 23 ml of growth medium.

Bacteria were introduced into the peripheral chamber of the model andallowed to grow for 2 hours prior to the first “dose” of drug. Drugdoses were administered into the central compartment and pumped toperipheral chambers by a peristaltic pump. Concentrations in the modeldeclined according to first order elimination (half-life) by dilution ofthe central compartment with drug free medium introduced by anadditional peristaltic pump adjusted to the desired clearance.

Samples (0.3 ml) were collected from peripheral compartments at variousintervals for determination of drug and bacterial concentrations.Samples were collected from the peripheral compartments and assayed fordrug concentrations by HPLC.

Bacterial Test Strains

Pseudomonas aeruginosa PAM1032 and PAM1582. The MICs of these strains tolevofloxacin were 1.0 and 32 ug/ml, respectively.

Inocolum Preparation

Strains were grown aerobically overnight in Mueller-Hinton Broth (MHB)at 35° C. and subcultured to fresh MHB and reincubated at 35° C. for 2hours. After 2 hours, the inoculum was further diluted 1:1000 to a finalconcentration of approx. 1.0×106 CFU/ml. Of the resulting dilution, 2.3ml was injected into each peripheral chamber of the hollow-fiberbioreacters (Unisyn, Hopkinton, Mass.).

Pharmacokinetics

The half-life of levofloxacin was adjusted to be 10 minutes to beequivalent to that observed following aerosol delivery of levofloxacinto the pulmonary compartment of man. The targeted Cmax was 1000 and 600ug/ml over two experiments.

Results

As targeted, the model exhibited a levofloxacin half-life of 10 minutesand the Cmax of 1000 ug/ml for Experiment 5. By comparison, Experiment 6was modified to achieve the same half-life as Experiment 5, but with atargeted Cmax of 600 μg/ml.

The bactericidal effects of these two regimens correlated with the Cmax.In Experiment 5 with a Cmax of 1000 ug/ml, the maximum bactericidaleffect was observed as a 5 log reduction in bacterial counts within 10minutes with PAM1032 and a 4 log reduction in bacterial counts within 20minutes with PAM1582 and no re-growth observed over the remaining 2hours of the experiment (FIG. 10). In contrast, while the Cmax of 600ug/ml used in Experiment 6 maintained the 5-log reduction in bacterialcounts for PAM1032, albeit taking 30 min instead of 10 min observed inExperiment 1, only a 3-log reduction in bacterial counts was observedfor PAM1582 after 45 min (FIG. 11). Moreover, PAM1582 exhibited initialre-growth before the end of the 2 hour experimental window.

Conclusions

Levofloxacin can produce up to a 99.9999% bacterial reduction with aCmax of both 600 and 1000 ug/ml against a strain with an MIC of 1 ug/ml.However, maximal bactericidal activity requires 3× more time at a Cmaxof 600 ug/ml. Levofloxacin can also produce up to 99.99% bacterialreduction with a Cmax of both 600 ug/ml against a strain with an MIC of32 ug/ml. However, the time to reach the maximum effect is 45 minutes.In contrast, levofloxacin can produce up to 99.999% bacterial reductionof this resistant strain with a Cmax of 1000 ug/ml and the time tomaximum effect is reduced to 20 minutes. From these results, extremelyhigh, but short duration exposures of levofloxacin produce rapid andsustained bacterial killing in both flask and hollow fiber models. Takentogether, the above results indicate that achieving an initial 800 ug/mllevofloxacin or other fluoroquinolone human ELF or sputum concentrationis sufficient to achieve the above antibiotic affects for the MIC99population as represented by PAM1582 (MIC of 32 ug/ml).

Example 2 Determination of the Aerosol Properties of AntibacterialFluoroquinolones

Introduction

Objective. The purpose of these studies was to evaluate the ability toformulate and deliver by nebulization a variety of fluoroquinolones fortreatment of pulmonary bacterial infections by aerosol administration.The fluoroquinolones evaluated are shown in Table 6.

TABLE 6 Fluoroquinolones Tested SP MSSA MRSA Pa Fluoro- MIC₉₀ MIC₉₀MIC₉₀ MIC₉₀ quinolone (ug/mL) (ug/mL) (ug/mL) (ug/mL) Approval StatusCiprofloxacin 2 1 64 8 Approved Gemifloxacin 0.06 0.06 2 8 ApprovedLevofloxacin 2 0.5 16 8 Approved Marbofloxacin 2 2 ND 8 VeterinaryGatifloxacin 0.5 0.125 4 16 Approved Ofloxacin 2 1 >32 16 ApprovedTosufloxacin 0.5 0.125 >16 16 Japan Lomefloxacin 16 2 >32 32 ApprovedMoxifloxacin 0.25 0.125 2 32 Approved Sparfloxacin 0.5 0.125 16 32Withdrawn Orbifloxacin 2 2 ND >32 Veterinary Pefloxacin 32 2 >32 >32Europe Trovafloxacin 0.25 0.06 8 >32 Withdrawn

These fluoroquinolones were chosen based on their availability, approvalstatus and antimicrobial properties. All tested fluoroquinolones areeither currently approved in the United States or have been approved butlater withdrawn due to various adverse reactions. In addition, severalfluoroquinolones, which are in use for veterinary applications, havealso been evaluated. Among bacterial pathogens responsible forrespiratory tract infections, Pseudomonas aeruginosa (Pa) andmethicillin resistant Staphylococcus aureus (MRSA) are the mostrefractory to treatment with fluoroquinolones. Streptocossus pneumonia(Sp) is probably the most important pathogen responsible for respiratorytract infections and numerous reports demonstrate high rates offluoroquinolone resistance in these bacteria. MIC₉₀ for Pa ranges from 4ug/ml to 32 ug/ml and from 2 ug/ml to >32 ug/ml for Pa and MRSA,respectively. Ciprofloxacin, levofloxacin, gemifloxacin and gatifloxacinvs gemifloxacin and moxifloxacin are the most potent against Pa andMRSA, respectively.

Table 7 contains a list of additional fluoroquinolones for potentialevaluation. The most microbiologically interesting compounds in the listare clinafloxacin and olamufloxacin, which were discontinued due toadverse reactions, and sitofloxacin, which is in Phase III clinicaltrials.

TABLE 7 Fluoroquinolones for Potential Evaluation Sp Sa MRSA Pa Fluoro-MIC₉₀ MIC₉₀ MIC₉₀ MIC₉₀ quinolone (ug/mL) (ug/mL) (ug/mL) (ug/mL) MarketStatus Clinafloxacin 0.06 0.06 2 4 Discontinued Sitafloxacin 0.06 0.1254 8 Phase III Olamufloxacin 0.06 1 2 16 Discontinued Norfloxacin 16 1 >416 Approved Prulifloxacin 1 0.25 32 16 Phase III Danofloxacin NA 0.125NA >16 Veterinary Enrofloxacin 1 0.125 8 >16 Veterinary Sarafloxacin NA0.25 >16 >16 Veterinary Balofloxacin 0.5 0.25 8 32 Korea Fleroxacin 81 >4 32 Europe Difloxacin 2 0.5 NA 32 Veterinary Rufloxacin 32 2 64 32Europe, China Enoxacin 16 1 >4 >32 Withdrawn Garenoxacin 0.06 0.06 8 >32Phase III Grepafloxacin 0.5 0.125 32 >32 Withdrawn Pazufloxacin 40.5 >16 >32 Japan

The fluoroquinolones in these two tables represent one field of optionsfor an aerosol fluroquinolone candidate. Several potent fluoroquinolonessuch as DX-619 and DW-286, which are at the early stage of clinicaldevelopment, might also be of interest for future studies.

Specific physico-chemical considerations for nebulization includeaqueous solubility, viscosity and surface tension. The aqueoussolubility of the drug should advantageously be sufficient to meet orexceed the minimal dosing requirement. The loading drug concentrationalso affects delivery time. Longer delivery times may be commerciallyunacceptable or lead to poor patient compliance. Although longerdelivery times may in effect modify the AUC shape, by non-limitingexample, the PARI eFlow device has been discovered to administer 4 ml ofaqueous levofloxacin in less than 5 min. Moreover, using such anefficient device, high concentration levofloxacin may be able to deliverthe effective doses described herein in a timeframe further enabling therapid administration, high concentration drug requirements needed foroptimal fluoroquinolone therapy.

In the case of fluoroquinolones, pH directly affects solubility. Ingeneral, solubility decreases significantly with increase in pH in therange 1.5 to 6.5. Because pH also affects patient tolerability (seebelow), the optimal choice of fluoroquinolone for pulmonary delivery viaaerosol has certain solubility and pH levels.

For the purpose of this feasibility study, the target solubility was setat 10 mg/mL or higher at a pH of 4.5 or greater, based on calculationsof therapeutic dose and the delivery metrics for available nebulizers.In order to be above the mutant prevention concentration (MPC), peakconcentration of fluoroquinolone after aerosol administrationadvantageously reaches about 100 ug/ml to about 1000 ug/ml at the siteof infection, pending the MIC of the infecting organism. Based on theseconsiderations, the minimal dose to be in this therapeutically relevantrange was projected to be at least about 30-40 mg Respirable DeliveredDose (RDD). Given the relative half-life of levofloxacin in the humanlung, the practical achievement of this dose by nebulization may beobtained with a loading dose of at least about 100 mg in a volume ofabout 2 mL (about 50 mg/mL) in a high-efficiency vibrating-mesh deviceoperating at its maximum performance efficiency delivering this dose inless than 4 min. A standard ultrasonic or jet nebulizer may require aloading dose of at least about 400 mg in a volume of about 5 mL (about80 mg/mL). However, the rate of administration by these less efficientdevices may not be sufficient to achieve high local concentration withshort duration exposure. Similar efficacious doses may also be achievedby administration of levofloxacin as a dry powder, where the rapidsolubility properties of levofloxacin may permit a quick dissolutionresulting in these desired soluble drug concentrations. However,alternative concentrations or alteration of the fluoroquinolone AUCshape profile may be desirable.

Alternatively, although aqueous solubility is important, it isreasonable to predict a formulation utilizing particle or complexationtechnology to enable nebulization of less soluble fluoroquinolones.Unfortunately, more intricate formulations increase both the complexityand cost of drug development, and in the case of jet and ultrasonicnebulizers, a significant reduction in efficiency of delivery, and limitthe ability to introduce other design elements into a final drugproduct.

In addition to drug solubility, for vibrating mesh devices nebulizationis also sensitive to the surface tension of the drug formulation.Therefore, in one embodiment, the surface tension is adjusted duringformulation by modifying drug concentration, excipient concentrationand/or the addition of surfactant.

In addition to factors that affect efficient nebulization, other factorsmay be considered for patient tolerability and compliance. Bynon-limiting example, these factors may include osmolality, pH andtaste. Osmolality affects acute tolerability in the respiratory tractand can be optimized for most drugs during formulation. Similarly, thepH of an aerosol also contributes to tolerability, yet only negativelywhen the formulation pH is less than 4.5. Therefore, because pH directlycontributes to fluoroquinolone solubility, fluoroquinolones that requirea pH less than 4.5 for solubility are likely to be poorly tolerated.Finally, fluoroquinolone taste can affect good patient compliance.Fluoroquinolones are known generally to be associated with anunpleasant, sometimes very intense taste. While there are technologiesavailable that may mask poor drug taste, these technologies increasedevelopment complexity and cost, and may not be totally effective in thecase of fluoroquinolones. Thus, similar to pH, taste may be consideredin identifying a fluoroquinolone suitable for nebulization.

Preparation and Characterization of the Test Solutions

Antibiotics were purchased from one of several sources as shown in Table8.

TABLE 8 Preparation of Fluoroquinolone Test Solutions Fluoro- Volume No.quinolone Source^(a) Purity^(b) Amount H₂O Final Conc. 1 GatifloxacinLKT 99.6  8.7 mg 0.87 mL 10 mg/mL 2 Gemifloxacin LG 99.6  9.5 mg 0.95 mL10 mg/mL 3 Levofloxacin LKT 99.2 10.3 mg 1.03 mL 10 mg/mL 4 MoxifloxacinLKT 99.5 12.5 mg 1.25 mL 10 mg/mL 5 Ciprofloxacin LKT 99.3 19.5 mg 1.95mL 10 mg/mL 6 Ofloxacin LKT 99.1 11.7 mg 1.17 mL 10 mg/mL 7 LomefloxacinMPI NA 17.0 mg 1.70 mL 10 mg/mL 8 Marbofloxacin Vetoquino NA  4.8 mg0.48 mL 10 mg/mL 9 Orbifloxacin MPI NA  4.2 mg 0.42 mL 10 mg/mL 10Pefloxacin MPI NA 15.0 mg 1.50 mL 10 mg/mL 11 Sparfloxacin MPI NA 14.5mg 1.45 mL 10 mg/mL 12 Tosufloxacin MPI NA 15.2 mg 1.52 mL 10 mg/mL 13Trovafloxacin MPI NA  2.0 mg 0.20 mL 10 mg/mL ^(a)LKT: LKT Laboratories.LG: LG Chem. NA. Source unavailable. ^(b)Purity of material tested.Described as GAR) or in percent API. c. 25 mg/ml solution

A 2 to 20 mg sample of each antibiotic was weighed into sterile plastictubes and brought up with a volume of sterile water to make a 10 mg/mLsolution or suspension of the antibiotic. Samples were incubated forapproximately 10 minutes at room temperature with occasional mixing,prior to further handling.

Following the incubation period, the antibiotic solutions were observedfor their visible appearance, with results as shown in Table 9.

Five of the fluoroquinolones tested were visibly soluble, and eithercolorless, or a shade of yellow. Eight were visibly insoluble, appearingeither cloudy (fine particulate), opaque (dense fine to mediumparticulate), or turbid (thick, large particulate slurry), in all caseswith a visible sediment. The pH of these initial solutions weredetermined, and ranged from 3.5 to 7.0. The insoluble solutions weretitrated with 1N HCl to the point of visible solubility, and the pH ofthe solubilized solution determined. In three cases, marbofloxacin,sparfloxacin and tosufloxacin, solubility was not reached by pH 1.5, andfurther addition of acid was stopped. With the exception of ofloxacin,the pH of these titrated solutions was in the range of 1.5 to 3.0.

TABLE 9 Fluoroquinolone Solution Characteristics After pH Adjustment 1NInitial Solution HCl No. Fluoroquinolone Appearance pH (uL)Appearance^(a) pH 1 Gatifloxacin white, cloudy, visible 7.0  5 slightyellow color, 3.0 sediment transparent, no sediment 2 Gemifloxacincolorless, transparent, no 4.7 NR — sediment 3 Levofloxacin slightyellow color, 4.7 NR — transparent, no sediment 4 Moxifloxacin brightyellow color, 4.7 NR — transparent, no sediment 5 Ciprofloxacin white,opaque (very dense), 5.5 60 colorless, transparent, no 2.0 visiblesediment sediment 6 Ofloxacin cloudy, visible sediment 6.5 10 slightlyyellow color, 5.2 transparent, no sediment 7 Lomefloxacin cloudy,visible sediment 4.2 — transparent, no sediment, — after 10 min. at rm.temp. 8 Marbofloxacin white, very turbid, visible 6.5 40 white, turbid,visible 1.5 sediment sediment 9 Orbifloxacin white, cloudy, visible 20colorless, transparent, no 1.7 sediment sediment 10 Pefloxacincolorless, transparent, no 4.5 NR — precipitate 11 Sparfloxacin brightyellow, turbid, visible 5.0 20 bright yellow, densely 1.5 sedimentturbid, visible sediment 12 Tosufloxacin white, turbid, visible 3.5 20white, cloudy, less turbid, 1.5 sediment visible sediment 13Trovafloxacin colorless, slightly cloudy, no 4.2 NR — sediment ^(a)NR:pH adjustment not required. Fluoroquinolone was solution at a pH >4 inthe initial solution.

After the pH adjustment, and following a further 10 minute incubationperiod with occasional mixing, the final appearance of the solutions wasdetermined, just prior to the aerosol tolerability and taste test.Results are shown in Table 10.

TABLE 10 Appearance of Fluoroquinolone Final Solution No.Fluoroquinolone pH Solubility Color Sediment Opaqueness 1 Gatifloxacin3.0 + C none none to very slight 2 Gemifloxacin + C none none to veryslight 3 Levofloxacin 4.7 + VLY none none 4 Moxifloxacin 4.7 + Y +/−none 5 Ciprofloxacin 2.0 + C none none 6 Ofloxacin 5.2 + LY +/− none 7Lomefloxacin 4.2 + C +/− none to very slight 8 Marbofloxacin 1.5 −− W ++++ 9 Orbifloxacin 1.7 + C none slight 10 Pefloxacin 4.5 + C none slight11 Sparfloxacin 1.5 −−− DY +++ ++++ 12 Tosufloxacin 1.5 −− W ++ +++ 13Trovafloxacin 4.2 + C + slight Y = yellow; LY = light yellow; VYL = verylight yellow; DY = dark yellow; C = colorless; W = white.

The compounds exhibiting preferred solubility for solutions suitable fordelivery by inhalation (10 mg/mL at a pH of 4.5 or above), werelevofloxacin, gemifloxacin, moxifloxacin, ofloxacin and pefloxacin.Levofloxacin, ofloxacin and moxifloxacin exhibited the bestsolubility/pH characteristics.

Taste and Tolerability Evaluation

Two evaluations were done to determine the suitability of thefluoroquinolone solutions with respect to taste and tolerability.

First, in an oral taste test the taste of a 20 uL portion of test samplewas determined in a single, healthy human volunteer by placing thematerial directly onto the center front part of the tongue. Taste wasthen monitored over a 1 minute period. This test was performed on theinitial solutions prepared as well as the final solutions following pHadjustment. Data are shown in Table 11.

TABLE 11 Oral Fluoroquinolone Taste Test Fluoro- No. quinolone InitialSolution Final Solution 1 Gatifloxacin moderate bitter unpleasant strongbitter taste, slightly aromatic unpleasant almond-like taste, strongaftertaste 2 Gemifloxacin very bitter unpleasant taste not performedwith strong aftertaste, all the way into throat 3 Levofloxacin slightchemical taste, not performed slightly bitter, slight almond-like taste4 Moxifloxacin moderate bitter-sweet not performed unpleasant taste,slightly aromatic 5 Ciprofloxacin sweet almond-like taste very strongbitter taste all the way into the throat 6 Ofloxacin bitter unpleasant,moderate bitter almond-like taste unpleasant, almond-like taste 7Lomefloxacin moderate to strong not performed almond-like taste, notvery unpleasant 8 Marbofloxacin bitter unpleasant moderate to strongalmond-like taste bitter unpleasant almond-like taste 9 Orbifloxacinstrong unpleasant taste very strong, very unpleasant bitter taste 10Pefloxacin strong bitter unpleasant not performed almond-like taste 11Sparfloxacin slight taste strong almond-like taste 12 Tosufloxacin mildto moderate strong almond-like almond-like taste taste 13 Trovafloxacinvery strong bitter not done unpleasant almond-like taste, strongaftertaste

Lowering of the pH generally had the effect of enhancing the tasteproperties of the solution. Gatifloxacin, gemifloxacin, ciprofloxacin,orbifloxacin and trovafloxacin were the least desirable in tastetesting. Of the fluoroquinolones tested, Levofloxacin was the onlyfluoroquinolone that was tolerable with respect to taste, at theconcentration tested. Lomefloxacin had a moderately strong almond-liketaste, and the taste was slightly unpleasant.

In the second test, the tolerability and taste of a small aerosol samplefrom a 0.5 ml aliquot of the test formulation was determined in a singlehealthy human volunteer, following nebulization in a PART eFlownebulizer (Table 12).

TABLE 12 Aerosol Fluoroquinolone Tolerability and Taste Test Fluoro- No.quinolone Aerosol Tolerability and Taste 1 Gatifloxacin moderate bitterunpleasant taste, mild cough sensation 2 Gemifloxacin strong unpleasantbitter taste, strong aftertaste, mild cough sensation 3 Levofloxacinchemical taste, somewhat bitter, mild cough sensation 4 Moxifloxacinmoderate bitter unpleasant taste, some cough, strong bitter aftertaste 5Ciprofloxacin very strong, bitter unpleasant taste, immediate coughing 6Ofloxacin bitter chemical taste, mild cough sensation 7 Lomefloxacinchemical taste, somewhat bitter, mild cough sensation 8 Marbofloxacintoo insoluble to test 9 Orbifloxacin very acidic, strong bitterunpleasant taste, strong cough 10 Pefloxacin chemical taste, some cough11 Sparfloxacin too insoluble to test 12 Tosufloxacin too insoluble totest 13 Trovafloxacin bitter unpleasant taste, no cough or coughsensation, no aftertaste

In the case of orbifloxacin, marbofloxacin and trovafloxacin, smallerportions were tested, due to solubility limitations. In a calibrationexperiment, the inhaler produced an aerosol output of 4.1 microns VIVID,with a geometric standard deviation (GSD) of 1.64 micron VIVID. Inaddition to these measurements, the inhaler produced a fine particledose (FPD) of 54.9% (percent of emitted dose in particles less than 5microns). The tolerability and taste of the drug during a very briefadministration period and for a period of 10 minutes post administrationwere monitored. Tolerability parameters were of the following types: (i)cough, cough sensation, or sneezing (ii) irritation, burning ortightness of throat, (ii) irritation or runniness in nasal passages oreyes, (iii) irritation, burning or tightness of the lungs or shortnessof breadth, and (iv) dizziness, headache, nausea or other systemiceffects.

Marbofloxacin, sparfloxacin and tosufloxacin were too insoluble toevaluate in this test. For the remaining fluoroquinolones tested, notolerability effects were observed during or after aerosol exposure incategories ii, iii or iv (above). Gatifloxacin, moxifloxacinciprofloxacin, orbifloxacin and pefloxacin were all associated withcough. In the case of ciprofloxacin and orbifloxacin this may have beenassociated with the low-pH of the solution. Of the fluoroquinolonestested, Levofloxacin at 10 mg/ml had the best taste characteristics.Ofloxacin, lomefloxacin and pefloxacin had a more discernible taste thanlevofloxacin, which were also acceptable during the short course ofadministration.

Summary and Conclusions from the Fluoroquinolone Taste Test

Of the thirteen flouroquinolones tested in this study, levofloxacin hadpreferred physical-chemical properties for aerosol administration and ademonstration of best acute tolerability of the fluoroquinolones tested(Table 13). Levofloxacin is also recognized as having one of the bestantimicrobial profiles for respiratory pathogens and has the highest invivo efficacy, comparable to ciprofloxacin, for treatment of Pseudomonasaeruginosa infections.

TABLE 13 Overall Suitability for Nebulization Fluoro- Overall No.quinolone Assessment Score Limitation 1 Gatifloxacin poor solubility andpH, moderately strong −− Solubility, Taste bitter aerosol taste 2Gemifloxacin sufficient solubility and pH, strong bitter −−− Tasteaerosol taste, strong aftertaste 3 Levofloxacin excellent solubility andpH, chemical + Taste aerosol taste, somewhat bitter 4 Moxifloxacinsufficient solubility and pH, moderately −− Taste, strong bitter aerosoltaste, strong aftertaste Pa Activity 5 Ciprofloxacin poor solubility andpH, very strong bitter −−− Solubility, Taste aerosol taste, coughing 6Ofloxacin minimally acceptable solubility and pH, −/+ Taste bitterchemical aerosol taste 7 Lomefloxacin minimally acceptable solubilityand pH, −/+ Pa Activity chemical aerosol taste, strong liquid taste 8Marbofloxacin very poor solubility even at low pH, −−− Solubility unableto test 9 Orbifloxacin very poor solubility even at low pH, −−−Solubility, strong bitter unpleasant aerosol taste, Taste, Activitystrong cough 10 Pefloxacin sufficient solubility and pH, aerosol −/+ PaActivity chemical taste, strong unpleasant liquid taste 11 Sparfloxacinvery poor solubility even at low pH, −−− Solubility, unable to test PaActivity 12 Tosufloxacin very poor solubility even at low pH, −−−Solubility unable to test 13 Trovafloxacin moderate solubility and pH,bitter aerosol −− Taste, taste Pa Activity

Ofloxacin, lomefloxacin and pefloxacin exhibited lower solubility andstronger taste at 10 mg/mL than levofloxacin. Ofloxacin is 2-fold lesspotent that levofloxacin, and lomefloxacin and pefloxacin are 4-foldless potent. Higher concentrations of these antibiotics have thepreferred potency and administration times under 15 minutes.

In a separate study, conducted in a similar manner, norfloxacin wastested and found to have a solubility, taste and potency profile verysimilar to gatifloxacin, with the exception of significantly lessactivity against the gram-positive pathogens.

Taste Testing of Aerosol Salt Formulations of Levofloxacin andGemifloxacin

Based on the results of the above studies, levofloxacin, and itsracemate ofloxacin, as well as gemifloxacin, and to a lesser extentgatifloxacin and norfloxacin are amenable to aerosol administration forpulmonary antibacterial treatment. To further test the taste and acutetolerability (cough sensation and cough) properties of levofloxacin andgemifloxacin, several formulations were prepared with different organicand inorganic acids and tested in the manner described above. Solutionswere prepared by first adding 500 mg levofloxacin to 10 ml of water oradding 500 mg of gemifloxacin to 20 ml of saline (due to solubilitylimitations), titrating the pH to ˜6.5 with HCl or organic acid, thenadjusting the osmolality of levofloxacin containing solutions to ˜300mOsmol/kg with sodium chloride. The formulations tested are shown inTable 14.

TABLE 14 Levofloxacin and Gemifloxacin Formulations Osmo- Conc lalityFluoro- (mg/ (mOsm/ quinolone Acid mL) pH kg) Levofloxacin HC1 50 6.5181 Levofloxacin Acetic 50 6.41 273 Levofloxacin Citric 50 6.45 286Levofloxacin Lactic 50 6.42 286 Levofloxacin Ascorbic 50 6.50 278Levofloxacin Tartaric 50 6.35 286 Gemifloxacin HC1 25 5.6 330Gemifloxacin Citric 25 5.7 363 Gemifloxacin Ascorbic 25 5.9 347

These formulations were tested by a total of three healthy humanvolunteers in the same manner as described above, at a levofloxacinconcentration of 50 mg/mL, and a gemifloxacin concentration of 25 mg/mL,in a carefully controlled, head-to-head, fully blinded test. Results areshown in Table 15 and 16.

TABLE 15 Aerosol Taste and Tolerability of Levofloxacin Formulation at50 mg/mL. Taster Acid 1 2 3 HC1 Moderate bitter Bitter taste, coughBitter taste taste sensation Acetic Acid Very acidic taste Strong acidictaste, Acidic taste, cough sensation after taste Citric Acid Mild aftertaste, Mild taste, sweet Mild after taste slightly sweet Lactic AcidStrong bitter taste, Mild taste, after Bitter, mild aftertaste taste,slight cough after taste Ascorbic Mild taste, slight Mild taste Littletaste or Acid acidity after-taste Tartaric Acid Very bitter, strongStrong bitter taste, Bitter taste after taste bitter after taste

TABLE 16 Taste and Tolerability of Gemofloxacin Formulatons at 25 mg/mL.Taster Acid 1 2 3 Hydrochloric Metallic taste, Slight bitter tasteBitter taste, Acid strong after taste slightly metallic Citric AcidSlightly sweet Mild cough, slightly Very mild taste, bitter no aftertaste Ascorbic Mild taste Cough, mild bitter Slightly bitter, Acid aftertaste mild after taste

These results demonstrate that hydrochloric acid, citric acid andascorbic acid formulations of levofloxacin have superior taste andtolerability compared to the acetic acid, lactic acid and tartaric acidformulations of levofloxacin. Furthermore, these levofloxacinformulations have superior taste and tolerability over the equivalentgemifloxacin formulations. With respect to gemifloxacin, the citric acidformulation had superior taste and tolerability compared to the HCl andascorbic acid formulations of gemifloxacin, and with further formulationrefinement, would be amenable to aerosol administration.

Taste Testing of Additional Aerosol Levofloxacin Formulations

To further test the taste and tolerability properties of additionallevofloxacin excipient combinations in a systematic manner, a series offormulations were prepared and tested. The formulations are listed inTable 17. They included sugars, salts, sweeteners and other excipientsprepared by mixing levofloxacin with water, adding the excipients listedin Table 17, and titrating if necessary to the desired pH with diluteHCl, Osmolality was not optimized for these studies. However, osmolalitywas determined using an Advanced Instruments Model 3250 Osmometer. Thismeasurement, made on 250 μL of sample, relies on freezing pointdepression to determine osmolality.

These formulations were tested in a total of three healthy humanvolunteers in a series of tests (A-G) in the same manner as describedabove, in a carefully controlled, head-to-head, fully blinded manner.All tests were performed in a fully blinded fashion. Results of thetests (Tables 19-25) are described below. The following scoring systemwas used (Table 18).

Test A: Taste Testing of Sweeteners, Divalent Metal Salts, and SurfaceActive Agents.

This test included sweeteners, calcium and magnesium salts, and surfaceactive agents (i.e., glycerin and PS-80). As shown in Table 17,formulations containing the sweeteners shown are mildly bitter and havemetallic taste. The artificial sweeteners appeared to produce a bittertaste that is distinct from the bitterness otherwise observed. Mostsignificantly, the formulation containing CaCl₂ had the most improvedtaste relative to control (MgCl₂ was not tested in this experiment)(Table 19).

Test B: Taste Testing of Mono- and Disaccharides in the Presence ofCalcium Chloride.

All of the formulzations screened in this experiment were well toleratedand tasted better than the control sample. Formulations containing boththe calcium salt and sugar performed better than either one alone,suggesting that these compounds improve taste through differentmechanisms. Of these formulations, 5% CaCl₂+7.5% glucose performed best.Note that the lactose is present at a lower concentration than the othersugars (Table 20).

TABLE 17 Levofloxacin Formulations Containing Various Excipients Osmo-Conc lality Fluoro- (mg/ (mOsm/ quinolone mL) Excipients pH kg)Levofloxacin 50 Control A (0.225% NaC1) 6.50 180 Levofloxacin 50Aspartame (0.1%) 6.49 175 Levofloxacin 50 Sucrulose (0.1%) 6.49 178Levofloxacin 50 Glucose (5%) 6.5 380 Levofloxacin 50 Sucrose (7.5%);NaCl (0.225%) 6.51 329 Levofloxacin 50 Glycerin (5%) 6.48 880Levofloxacin 50 PS-80 (0.1%) 6.51 189 Levofloxacin 50 CaCl₂ (5%) 6.10784 Levofloxacin 50 MgSO₄ (5%) 6.41 73 Levofloxacin 50 Control - B-E(0.225% NaC1) 6.51 182 Levofloxacin 50 CaCl₂ (5%) 6.1 735 Levofloxacin50 CaCl₂ (5%), Sucrose (7.5%) 6.10 958 Levofloxacin 50 CaCl₂ (5%),Glucose (7.5%) 6.10 1174 Levofloxacin 50 CaCl₂ (5%), Glucose (7.5%) 5.251246 Levofloxacin 50 CaCl₂ (5%), Lactose (5%) 6.07 864 Levofloxacin 50MgCl₂ (5%) 5.90 600 Levofloxacin 50 MgCl₂ (5%), Sucrose (7.5%) 5.98 815Levofloxacin 50 MgCl₂ (5%), Glucose (7.5%) 5.98 999 Levofloxacin 50MgCl₂ (5%), Glucose (7.5%) 5.04 1035 Levofloxacin 50 MgCl₂(5%), Lactose(5%) 5.96 697 Levofloxacin 50 MgSO₄(5%), Sucrose (7.5%) 6.20 433Levofloxacin 50 MgSO₄(5%), Glucose (7.5%) 6.21 625 Levofloxacin 50MgSO₄(5%), Glucose (7.5%) 5.40 660 Levofloxacin 50 MgSO₄(5%), Lactose(5%) 6.18 387 Levofloxacin 50 Control F- G (0.45% NaC1) 6.5 221Levofloxacin 50 Glucose (5%) 6.5 376 Levofloxacin 50 Sucrose (5%) 6.5240 Levofloxacin 50 Lactose (5%) 6.62 241 Levofloxacin 50 Lactose (2.5%)6.55 170 Levofloxacin 50 CaCl₂ (5%) 6.10 735 Levofloxacin 50 CaCl₂ (5%),Lactose (5%) 6.21 1037 Levofloxacin 50 CaCl₂ (2.5%), Lactose (5%) 6.36565 Levofloxacin 50 CaCl₂ (2.5%), Lactose (2.5%) 6.41 370 Levofloxacin50 CaCl₂ (1.25%), Lactose (2.5%) 6.64 227 Levofloxacin 50 CaCl₂(0.625%), Lactose (2.5%) 6.06 163

TABLE 18 Taste Test Scoring System. Score Taste Tolerability 1Comparable to saline No cough sensation, no cough 1.25 Slightly moretaste than saline Slight cough sensation, no cough 1.5 Mildbitter/metallic taste Cough sensation, slight cough 1.75 Between 1.5 and2 — 2 Moderate bitter/metallic taste Cough sensation, moderate cough2.25 Between 2 and 2.5 — 2.5 Strong bitter/metallic taste — 2.75 Between2.5 and 3 — 3 Very strong bitter/metallic taste Cough sensation andstrong cough 4 Very strong bitter/metallic taste Cough sensation, strongcough and other unacceptable taste and other irritation

TABLE 19 Taste and Tolerability of Levofloxacin Formulations ContainingSweetners, Divalent Metal Salts, and Surface Active Agents Taster 1 2 3Median Excipients Taste Tol. Taste Tol. Taste Tol. Taste Tol. Aspartame(0.1%) 2 1.25 2 1 2 1 2 1 Sucrulose (0.1%) 2 1 1.75 1 2 1 2 1 Sucrose(7.5%); NaCl (0.225%) 2 1 2.25 1 2 1 2 1 Glucose (5%) 1.5 2 2.5 1 2 1 21 Glycerin (5%) 2.25 1 2.25 1 2.5 1 2.3 1 PS-80 (0.1%) 1.75 1 2.25 1 2.51 2.3 1 CaCl₂ (5%) 1.25 1 1.5 1.5 2 1 1.5 1 MgS0₄ (5%) 1.5 1.5 2.5 2.52.5 1 2.5 1.5 Control-A (0.225% NaCl) 3 1 3 1 2.5 1 3 1

TABLE 20 Taste and Tolerability of Levofloxacin CaCl₂ FormualationsTaster 1 2 3 Median Excipients Taste Tol. Taste Tol. Taste Tol. TasteTol. CaCl₂ (5%) 1.75 1 2 1 2.75 1 2 1 Sucrose (5%) 2 1 2 1 2 1 2 1 CaCl2(5%)₂, Sucrose (7.5%) 1.75 1 1.75 1 1.5 1 1.8 1 CaCl₂(5%), Glucose(7.5%) 1.5 1 1.5 1 2 1 1.5 1 CaCl₂(5%), Lactose (5%) 1 1 1.75 1 2 1 1.81 Control B-E (0.225% NaCl) 3 1 2.5 1 3 1 3 1

Test C: Taste Testing of Mono- and Disaccharides in the Presence ofMagnesium Chloride.

As above, all of the formulations screened in this experiment were welltolerated and tasted better than the control sample. Formulationscontaining both the magnesium salt and lactose appeared to performslightly better than either one alone. This experiment confirms thatcombining divalent metal salts and simple sugars are effective atimproving taste (Table 21).

TABLE 21 Taste and Tolerability of Levofloxacin MgCl₂ FormulationsTaster 1 2 3 Median Excipients Taste Tol. Taste Tol. Taste Tol. TasteTol. MgCl₂ (5%) 1.5 1 — — 1.75 1 1.6 1 MgCl₂ (5%), Sucrose (7.5%) 1.5 11.75 1 2 1 1.8 1 MgCl₂(5%), Glucose (7.5%) 1.25 1 2.25 1 2 1 2 1 MgCl₂(5%), Lactose (5%) 1 1 1.5 1 1.5 1 1.5 1 Control B-E (0.225% NaC1) 2.251 — — 2.75 1 2.5 1

Test D: Taste Testing of Mono- and Disaccharides in the Presence ofMagnesium Sulfate.

As with calcium and magnesium chloride, formulations containingmagnesium sulfate and glucose, sucrose or lactose tasted better than thecontrol sample. This experiment reconfirms that combining divalent metalsalts and simple sugars improve taste (Table 22).

TABLE 22 Taste and Tolerability of Levofloxacin MgSO₄ FormulationsTaster 1 2 3 Median Excipients Taste Tol. Taste Tol. Taste Tol. TasteTol. MgSO₄, Sucrose 1.5 2 1.5 1.25 1.5 1 1.5 1.3 MgSO₄, Glucose 1.5 2.752 2.5 1.5 1.5 1.5 2.5 MgSO₄, Lactose 1.25 2.25 1.75 1.25 1.75 1 1.8 1.3Control B-E (0.225% NaC1) 2.25 1 — — 3 1 2.6 1

Test E: Taste Testing of Divalent Metal Salts in the Presence of Glucoseat Low and High pH.

In this experiment, the effect of glucose in combination with each ofthe three divalent cation salts on taste and tolerability was tested atlow (≦5.5) and high (≧6.0) pH. Small but consistent improvements intaste were observed at the higher pH (Table 23).

TABLE 23 Taste and Tolerability of Levofloxacin CaCl2 Formulations atLow Versus High pH Taster 1 2 3 Median Excipients Taste Tol. Taste Tol.Taste Tol. Taste Tol. CaCl₂ (5%), Glucose (7.5%), pH6.1 1 1 1.5 1 2 11.5 1 CaCl₂ (5%), Glucose (7.5%), pH 5.5 1.25 1 1.75 1 2.5 1 1.8 1 MgCl₂(5%), Glucose (7.5%), pH 6.0 1.25 1 2 1 2 1 2 1 MgCl₂ (5%), Glucose(7.5%), pH 5.0 1.75 1 1.75 1 1.5 1 1.8 1 MgSO₄ (5%), Glucose (7.5%), pH6.2 1.25 2.25 2.25 1.75 1.5 1 1.5 1.8 MgSO₄ (5%), Glucose (7.5%), pH 5.41.5 1.75 1.75 1.5 2 1 1.8 1.5 Control B-E (0.225% NaC1) 2 1 — —

Test F. Taste Testing of Mono- and Disaccharides.

All of the formulations screened in this experiment were well toleratedand tasted better than the control sample. All three sugars at 5% werebetter than the control, lactose at 2.5% tasted better than the control,but not as good as at 5%. This experiment reconfirms that simple sugarsimprove taste (Table 24).

TABLE 24 Taste and Tolerability of Levofloxacin Sugar FormulationsTaster 1 3 Median Excipients Taste Tol. Taste Tol. Taste Tol. Glucose(5%) 1.5 1.5 2 1 1.8 1.3 Sucrose (5%) 1.5 1.5 1.5 1 1.5 1.3 Lactose (5%)1.75 1.25 2 1 1.9 1.1 Lactose (2.5%) 2.25 1.5 2 1 2.1 1.3 Control F-G(0.45% NaC1) 2.5 1 2.5 1 2.5 1

Test G. Taste and Tolerability of Levofloxacin CaCl2 Formulations in thePresence of Lactose.

In this experiment, levofloxacin was formulated with varyingconcentrations of calcium chloride and lactose (Table 25). As notedthrough this series of experiments, all formulations containing divalentmetal salts and sugar were improved with respect to taste andtolerability relative to the control formulation. Most importantly, 5%calcium chloride or 2.5% calcium chloride in the presence of 5% lactosewere most effective at decreasing levofloxacin bitterness. Furtherdecreases in the concentration of these excipients were less effective.

TABLE 25 Taste and Tolerability of Levofloxacin CaCl2 Formulations inthe Presence of Lactose Taster 1 3 Median Excipients Taste Tol. TasteTol. Taste Tol. CaCl₂ (5%) 1.25 1 1.5 1 1.4 1 CaCl₂ (5%), Lactose (5%)1.25 1 2 1 1.6 1 CaCl₂ (2.5%), Lactose (5%) 1.25 1 2 1 1.6 1 CaCl₂(2.5%), Lactose (2.5%) 1.5 1 2.5 1 2 1 CaCl₂ (1.25%), Lactose 1.75 1 2 11.9 1 (2.5%) CaCl₂ (0.625%), Lactose 1.75 1.25 2 1 1.9 1.1 (2.5%)Control F-G (0.45% NaC1) 3 1 2.5 1 2.8 1

Example 3 Aerosol Levofloxacin Characterization in PARI LC Plus JetNebulizer

The following studies describe the potential for aerosolized delivery oflevofloxacin to be administered to a patient via a jet nebulizer. Toaccomplish this task, a simple levofloxacin formulation was prepared andthe aerosol was characterized in a jet nebulizer. The results of thesestudies are shown in the summary below.

Levofloxacin inhalation solution (55 mg/ml) was evaluated using a PARTLC Plus Air Jet Nebulizer with ProNeb Compressor. The emitted dose,particle size distribution and fine particle fraction were measured bycascade impaction using a Marple Miller Impactor. The above-mentionedparameters were used for evaluating the in vitro performance ofaerosolized medications.

Marple Miller Study

Objective.

To determine the particle size distribution and estimate the amount ofdrug that a patient is likely to inhale (respirable fraction). Asecondary objective was to estimate emitted dose, which is the amount oflevofloxacin that exited the nebulizer.

Methods.

Formulation: 55 mg/ml levofloxacin, 120 mM chloride, 70 mM sodium, pH6.7. Formulation established from maximum solubility permitting a 300 mgdosage in 6 ml and neutral pH. 5.5 ml of levofloxacin formulation wasadded to a PART LC Plus Air-Jet Nebulizer with ProNeb Compressor. Thenebulizer cup contained a total of 302 mg of levofloxacin. The nebulizerwas connected inline with a Marple Miller Impactor (MMI), which was runwith an airflow rate of 60 l/min. Each nebulizer (n=2) was run todryness (no aerosol produced as judged by visual inspection for 15minutes. Following aerosolization, the MMI was disassembled andlevofloxacin was quantitatively extracted with mobile phase (90/10ACN:water) from the USP entry port, each of the impactor collection cups(stages) and the glass fiber filter. Any remaining formulation in thenebulizer after aerosolization (cup and mouthpiece) was also quantified.

Results

As shown in Table 26, the total average amount recovered from the MMIexperiments was 170.2 mg. The expected recovery was 302 mg. Thisrepresents a total recovery of ˜57%, which does not meet the generallyaccepted specifications for impaction-based studies (85%-115% totalrecovery). This difference was found to be due to non-specific adherenceof levofloxacin to the LC Plus nebulizer device. The average percent ofdrug exiting the nebulizer in fine particles was ˜72%. Thus, therespirable emitted dose was 89.7 mg. Assuming that ˜50% is not inhaledduring normal tidal breathing, a total of −40 mg may be deposited in thelung with this 300 mg dose. However, given the slow administration timewith this device, competition with pulmonary clearance would likelyprevent the accumulation of sufficient levofloxacin to meet the requiredminimal concentration for “rapid administration, high concentration”dosing needed for maximum fluoroquinolone antimicrobial activity andresistance prevention.

TABLE 26 Marple Miller Impactor Data Set B A + B Percent of Total AAmount of Drug Amount Drug Exiting Emitted A Remaining in of DrugNebulizer in Fine Dose Nebulizer Cup Recovered Particle Fraction SampleID (mg) (mg) (mg) (% < 5 um) Levo 134.70 45.00 179.70 73.5 Run 1 Levo114.40 46.30 160.70 70.4 Run 2 Average 124.6 45.7 170.2 72.0

Example 4 Animal Models and Evaluation of Fluoroquinolones andFluoroquinolone Formulations

Pharmocokinetic Model

Six rats per study are given a single slow bolus intravenous dose of 10mg/kg via the lateral tail vein or are given a single microspray aerosoldose of 10 mg/kg using a microspray aerosol generation device(PennCentury, Philadelphia, Pa.). Blood samples are taken at varioustimes over 3 hours to determine the plasma pharmacokinetic parameters.Two rats are sacrificed at 0.5, 1.5 and 3 hours after dosing todetermine lung, broncheoalveolar lavage (BAL), and epithelial liningfluid (ELF) levels. The plasma and tissue concentrations are determinedby an HPLC method and the data are then fit using WinNonlin. Data areshown in Table 27.

TABLE 27 Pharmacokinetic Modeling ELF Dose Serum AUC F, % from DrugRoute (mg/kg) AUC (0-inf) Serum t½ (0.5-3 h) Lung vs. IV Levofloxacin IV10 3.8 0.5 10.5 NA Levofloxacin IT 10 3.28 0.4 12.07 86% CiprofloxacinIV 10 2.56 0.53 ND NA Ciprofloxacin IT 3.3 0.8 0.93 194 82%Clinafloxacin IT 10 3.2 0.74 30.8 Gatifloxacin IV 10 5.31 1.06 5.32Gatifloxacin IT 10 5.83 1.13 54.7 100% Norfloxacin IV 10 4.65 1.21 3.27Norfloxacin IT 10 4.46 1.13 41.7 100% Gemifloxacin IV 8 4.54 1.04 3.72Gemifloxacin IT 10 5.86 1.68 536.5 86% Tobramycin IV 10 15.7 0.5 27.6 NATobramycin IT 10 13.82 1.0 5152.0 81%

Efficacy Model

P. aeruginosa strain PAM 1723 is grown in Mueller-Hinton Broth (MHB) at35° C. under constant aeration, after 16 hours, the inoculum issub-cultured into fresh MHB and allowed to regrow at 35° C., underconstant aeration, for 4 hours. The inoculum is adjusted to ca. 5×10⁶CFU/ml by correlation of absorbance at 600 nm with predetermined platecounts. Male CFW mice (4-6 weeks old, N=4/group) are made neutropenic bythe intraperitoneal injection of 150 mg/kg cyclophosphamide (Cytoxan,Mead Johnson, Princeton, N.J.) on days 1 and 4. On day 5, mice areinfected by an intratracheal instillation of 0.05 ml of inoculum whileunder isoflurane anesthesia (5% isoflurane in oxygen running at 4L/min). Two hours after infection, mice are given either intraperitonealor intratracheal doses of each fluoroquinolone at a dose of 25 mg/kg.Mice are sacrificed 1 and 4 hours after treatment, their lungs removed,homogenized and plated to determine colony counts. Data are shown inTable 28.

TABLE 28 Efficacy Modeling Dose DeltaLOG DeltaLOG Drug Route^(a) (mg/kg)CPU 1 hr^(b) CPU 4 hr^(b) Levofloxacin IP 25 −1.00 −0.52 Levofloxacin IT25 −1.97 −1.28 Gemifloxacin IP 25 −0.28 −0.32 Gemifloxacin IT 25 −2.45−1.81 Levofloxacin IP 25 −1.40 −1.14 Levofloxacin IT 25 −2.48 −1.45Gemifloxacin IP 25 −0.74 −0.71 Gemifloxacin IT 25 −3.20 −2.28Clinafloxacin IP 25 −1.32 −1.33 Clinafloxacin IT 25 −2.86 −2.47Tobramycin IP 5 −0.70 0.29 Tobramycin IT 5 −1.59 −0.94 Ciprofloxacin IP25 −1.59 −0.41 Ciprofloxacin IT 25 −2.32 −1.45 Gatifloxacin IP 25 −0.34−0.02 Gatifloxacin IT 25 −1.48 −2.11 Clinafloxacin IP 10 −0.96 −1.39Clinafloxacin IT 10 −2.71 −2.40 Sparfloxacin IP 25 −0.85 0.09Sparfloxacin IT 25 −1.56 −0.81 Tosufloxacin IP 25 0.00 1.33 TosufloxacinIT 25 −0.48 −0.24 ^(a)Route of drug administration ^(b)Time post-drugadministration

In rat pharmacokinetic studies, aerosol administration offluoroquinolones results in increased ELF AUCs from 0.5-3 hours for allfluoroquinolones tested, as well as tobramycin, suggesting that theaerosol route of administration will produce increased efficacy againstlung infections.

In a mouse lung infection model, the increased efficacy, suggested bythe pharmacokinetic studies rats, was confirmed. For allfluoroquinolones tested, the aerosol route of administration(intratracheal, or IT), produced larger reductions in bacterial countsthan the intraperitoneal (IP) route of administration, suggesting thatobserved increased efficacy was due to high local concentrationsproduced by direct aerosol administration.

Example 5 Aerosol Levofloxacin Characterization in the PART eFlowNebulizer

Laser Particle Sizing

Device performance was characterized by measuring the size of theparticles emitted. By non-limiting example, particle sizing of emittedaerosol of Levofloxacin solution may be conducted with a MalvernSpraytec particle sizer under the following conditions. Ambientconditions are controlled to maintain a room temperature of between23.0° C. and 24.0° C. and relative humidity of 42% to 45%. Levofloxacinat 25 mg/ml was loaded into 2 PART eFlow Nebulizers fitted with the “40”nebulizing heads. Software for the Malvern Spraytec particle sizer isprogrammed to calculate the following information. A) Volume MeanDiameter (VIVID), the volume mean of the particles passing across thebeam of the laser. B) Geometric Standard Deviation (GSD), diameter84^(th) percentile/diameter 50^(th) percentile. C) % of particles ≦5microns, the percent of the number of particles less than 5 microns or %of particle >1 micron and <7 microns, the percent of the number ofparticles between 1 and 7 microns.

The device was loaded with 2 ml Levofloxacin at 25 mg/ml. The mouthpieceof the device was positioned with the tip of the mouthpiece 2 cm fromthe center of the beam on the x axis and as close to the optical lens ofthe laser as possible on the y axis. Ambient conditioned, bias flow wasprovided through the nebulizer in an amount to obtain a total nebulizerflow of 20 LPM. Ambient conditioned, bias flow was provided through thenebulizer in an amount to obtain a total nebulizer flow of 20 LPM. Thenebulizer was turned on and allowed to run continuously for 1 minuteprior to measuring. The measurement sequence was begun after 1 minuteand measurements are made continuously for 1 minute in 1 secondintervals. At the end of the measurement phase, these 60 records areaveraged for VIVID, GSD and %≦5 micron and %>1 and <7 micron. Finally,the nebulizer was weighed for determination of output rate.

Breath Simulation Studies

Device performance was measured under conditions similar to naturalinhalation by using a breath simulator PART Compas breath Simulatorprogrammed to use the European Standard pattern of 15 breaths per minutewith an inspiration to expiration ratio of 1:1. Such measurements wasperformed under ambient conditions that can be controlled to maintain aroom temperature of between 23.0° C. and 24.0° C. and relative humidityof 42% to 45%. For this experiment, the PART eFlow device was loadedwith 4 ml Levofloxacin solution at 25 mg/ml.

Breathing simulation was commenced, and the nebulizers begun. Thedevices were allowed to run continuously until nebulization ceases. Theduration is timed from the beginning of nebulization. Followingnebulization, the inspiratory and expiratory filters were individuallywashed in a known amount of solvent (dH₂O). The nebulizer cup is alsowashed individually. For quantitation, the individual washings wereassayed via spectrophotometry at a wavelength of 290 nanometers and theresultant concentration converted to content. Using this quantitativedata, the following analysis was made. A) Inspired dose (ID), the totalamount of drug assayed from the inspiratory filter. B) Residual dose(RD), the amount of drug assayed from the nebulizer at the end ofnebulization. C) Fine Particle Dose (FPD), the ID multiplied by therespirable fraction (for example, % particles ≦5 microns VIVID dependingon the method used to determine the size of the particles emitted fromthe selected device). D) Duration, time from the beginning to the end ofnebulization. E) Respirable Delivered Dose (RDD), % ID that is, forexample, ≦5 microns VIVID.

The results in Table 29 indicate that a 100 mg dose of levofloxacinlikely deposits ˜34 mg fluoroquinolone in the pulmonary compartment in˜4 min using the PART eFlow device (Table 29) compared to the 300 mgdose from the PAR LC Plus device delivering an equivalent dose in >15min. From the “rapid administration, high concentration” dosing anddelivery model described herein, while the 15 min delivery time from theLC Plus will likely fail, a 4 min administration time fo 35-40 mglevofloxacin may meet the criteria for maximum fluoroquinolone activity.However, increasing the drug concentration to enable more rapidadministration (e.g. 50 mg/ml in a 2 ml dosing delivering 35-40 mglevofloxacin in ˜2 min) will more likely meet these minimalrequirements. Moreover, shorter administration times will improvepatient dosing compliance. In addition, it should be noted thathypotonic solutions of levofloxacin at concentrations greatere than 10mg/ml are poorly tolerated for inhalation.

TABLE 29 Levofloxacin Aerosol Properties (100 mg Loading Dose) DurationResidual Inspired FPD (%) RDD (mg) VMD GSD Osmo (minutes) Dose Dose ≦5u1-7u ≦5u 1-7u um um mOs/kg 3.9 ± 0.1 24.8 ± 3.4 61.1 ± 1.6 54.9 73.833.5 45.1 4.7 1.6 67 ± 1.0

Example 6 Tolerability of Aerosol Levofloxacin in a Healthy HumanSubject

Methods

In a single subject, healthy volunteer the feasibility of deliveringlevofloxacin as an aerosol was established using either an AerogenClinical vibrating mesh device creating a 3.4 micron volumetric meandiameter (VIVID) particle, or ˜2 micron MMAD (hereinafter “AerogenSmall”), or using a PARI eFlow nebulizer producing a ˜4.7 micron VIVIDparticle (hereinafter “PARI Large”). Levofloxacin was tested at aconcentration of 4.25 mg/mL or 18.75 mg/mL at doses of 10 mg, 35 mg and55 mg, in isotonic solution.

Results

In the first test, 6 mL of the 4.25 mg/mL solution was inhaled using theAerogen Small nebulizer. The estimated RDD based on separate in vitrodevice characterization studies using breath simulation was estimated tobe 10 mg. Delivery time was 22 minutes. No discernable adverse effectswere observed in the throat, airway or lungs, during or afteradministration, including cough sensation or cough, and there was only aslight chemical taste during and after administration. No adverseeffects or taste were observed over a 30 minute monitoring periodfollowing drug administration. At this low concentration and dose, andslow rate of administration, levofloxacin was well tolerated.

In the second test, 4 ml of the 18.75 mg/mL solution was inhaled usingthe Aerogen Small nebulizer. The estimated RDD based on separate invitro device characterization studies using a breath simulator was 35mg. Delivery time for administration of the drug was 14 minutes. Despitethe increased dose, the acute tolerability was very comparable to thefirst test both during and after administration. The taste, which wasstronger, was the solution had a more the bitter/metallic chemical tastecharacteristic of levofloxacin. The taste was most discernible for aperiod of a few minutes after the end of administration, again acharacteristic of levofloxacin.

In the third test, 4 mL of the 18.75 mg/mL solution was inhaled usingthe PARI Large device. The estimated RDD based on separate in vitrodevice characterization studies was ˜55 mg (using the <5 microns FPDdefinition). Delivery time for administration of the drug was ˜5minutes. Despite the significantly increased particle size and deliveryrate for drug compared to test 2, no adverse effects in the throat,airway or lungs, other than the acute effects of taste noted above, wereexperienced, including cough sensation or coughing, throughout thedosing period and for a 30 minute observation period following deliveryof the dose. Urinary recovery of the drug, which is an accurate measureof exposure, confirms that the projected respirable dose ofapproximately 55 mg was successfully delivered.

These results demonstrate the feasibility of aerosol delivery oflevofloxacin in a human subject at the intermediate concentrationstested, and suggest that higher concentrations and doses, properlyformulated for tolerability and taste are achievable.

Example 7 Levofloxacin Micronization

Dry powder levofloxacin base may be micronized for high localconcentration exposure therapy, taste-masking or AUC shape-enhanceddelivery of levofloxacin using dry powder pulmonary administration.Other approaches currently being investigated include spray-dry and insitu micronization techniques. This approach may also be used with otherfluoroquinolone antibiotics including, without limitation ofloxacin,lomefloxacin, pefloxacin, ciprofloxacin, gatifloxacin, gemifloxacin,moxifloxacin, tosufloxacin, pazufloxacin, rufloxacin, fleroxacin,balofloxacin, sparfloxacin, trovafloxacin, enoxacin, norfloxacin,clinafloxacin, grepafloxacin, sitafloxacin, marbofloxacin, orbifloxacin,sarafloxacin, danofloxacin, difloxacin, enrofloxacin, garenoxacin,prulifloxacin, olamufloxacin, DX-619, TG-873870 and DW-276.

Description

To characterize the feasibility of micronizing levofloxacin base, thefollowing studies were performed.

Micronization

Levofloxacin drug powder was micronized using a jet mill. Followingmicronization the drug powder was collected in two fractions, onebetween 5-6 micron and a finer fraction.

Powder Characterization

The drug was characterized for particle size and particle sizedistribution, before and after milling using laser diffractiontechnology. Any changes in the physical form of the drug were evaluatedby Differential Scanning calorimetery (DSC) and X-Ray diffraction ()M)). Particle morphology was studied using Scanning Electron Microscopy(SEM). The equilibrium moisture content of the drug powder before andafter micronization was determined by Thermogravimetric Analysis (TGA)or Karl Fischer. Any degradation of the drug substance duringmicronization was evaluated by HPLC. The separation conditions were usedto determine if any new peaks are formed after micronization.

Micronization

Experimental Methodology

Two batches of Levofloxacin were micronized using a jet mill (GlenMills). Method development was performed to determine the micronizationpressure required to achieve the required size fractions between a) 5-6micron and b) 2-3 micron. The particle size of levofloxacin wasdetermined by Sympatec HELOS laser diffraction particle size analyzer.

Results

FIG. 12 shows the plot of mean particle diameter (X50) Vs micronizationpressure. The first batch of Levofloxacin showed a mean particlediameter of 10.6 micron before micronization. From the plot it is seenthat particle size decreased as the micronization pressure increased. Apressure of about 120 psi was required to achieve a size of 2.5 micron.With the second batch of levofloxacin, having a mean particle size of12.99 micron before micronization, a pressure of 30 psi was required toachieve a particle size of 5.2 micron.

Powder Characterization

Differential Scanning calorimetry

Experimental Methodology

Differential Scanning calorimetry of pre-micronized as well asmicronized levofloxacin (mean particle size 2.5 micron) was performedusing TA Instrument DSC Q1000. 1-2 mg of each sample was weighed intopan, sealed and heated at 10° C./min from 25° C. to 300° C. undernitrogen.

Results

The DSC profiles of the pre-micronized and micronized levofloxacin areshown as FIGS. 13A and 13B. There was no difference in the DSC profilesof micronized compared to pre-micronized levofloxacin.

Experimental Methodology

The powders (micronized and pre-micronized) were adhered to double-sidedcarbon tabs on aluminum stubs, which were then coated withgold-palladium. Photomicrographs were taken of several different areasof the powder on the stub using a Scanning electron microscope.

Results

Representative Scanning electron micrographs of the pre-micronized andmicronized levofloxacin are shown in FIGS. 14A and 14B. Crystals oflevofloxacin are plate like before micronization. This shape is retainedafter micronization.

Experimental Methodology

A thin layer of powder sample was mounted on a zero background plate inan XRD sample holder. Each sample was analyzed using a Scintag XDS 2000Diffractometer under the following conditions: Excitation source: CopperK a X-rays; Scan rate: 1° per minute; Voltage: 40 KV; Curent: 35 mA.

Results

The X-ray diffraction plots of pre-micronized and micronizedLevofloxacin are shown as FIGS. 15A and 15B. Intensity of diffractedpeak at 9° is reduced after micronization. These results are inaccordance to those reported in the literature for micronization ofolanzapine (Stephenson G. A. The Rigaku Journal, 22 (2005): 2-15). Thereduction in the relative intensities of the diffracted peaks might bedue to formation of new faces to a crystal. The most developed faceafter micronization would be the one for which the intensity ismaximally reduced.

Experimental Methodology

15-25 mg of micronized and pre-micronized levofloxacin samples weredissolved in methanol (having predetermined moisture content) and themoisture content in the samples were determined by Aquastar 3000Coulometric Karl Fisher Titrator.

Results

The results of Karl Fisher analysis are shown in Table 30.

TABLE 30 Moisture Content of Pre- and Post-Micronized LevofloxacinMoisture content (%) Pre- micronized 6.16 Post- micronized 5.42

Example 8 Preformulation of Levofloxacin Base

The goal of this study was to characterize levofloxacin base tounderstand the physico-chemical capabilities and restrictions oflevofloxacin base for various formulation approaches. The purpose ofthis study was to characterize the physicochemical properties oflevofloxacin base.

Preformulation

pH-Solubility Studies

The solubility of levofloxacin was determined as a function of pH.Buffers were first prepared in the pH range 2-10. Small aliquots of eachbuffer (˜200-250 μL) were saturated with drug and agitated to achieveequilibrium solubility. The samples were then becentrifuged and thesupernatant analyzed for dissolved drug by UV or HPLC. The buffers usedin this study were shown to affect the solubility result (becausedifferent buffer counter-ions can form different levo salt forms insolution). Hence, pH-solubility will also be assessed in the absence ofbuffers (via titration).

pKa Determination

The pKa of levofloxacin was determined by titrimetry. Obtained pKavalues were confirmed by UV spectrophotometry. This information was usedto aid in salt selection for levofloxacin and to determine the charge onlevofloxacin under the pH conditions in the lung.

Preformulation for Liquid System

The feasibility of a liquid formulation was investigated using (a)solubility and (b) surface tension as baseline parameters forformulation in saline alone.

Preformulation Studies on Levofloxacin

HPLC Method Transfer

Experimental Methodology

A HPLC method was used to evaluate the linearity, accuracy and precisionof the levofloxacin assay. The column used was a 50×4.6 mm, OnyxMonolithic C18 (Phenomenex) at 30o C. The mobile phase consisted of 85%of 0.1% TFA in water and 15% 0.1% TFA acetonitrile. The flow rate wasadjusted to 3 ml/min. Samples were injected into the chromatographicsystem and the effluent monitored at 277 nm.

Results

The retention time for levofloxacin was approximately 0.82 min. Theassay was found to be linear over a range of 5-15 μg/ml, with acorrelation coefficient of 1.000. RSD (relative standard deviation) wasless than 0.5% and accuracy was within 98-102%.

pH-Solubility Studies

By Titration

Experimental Methodology

A saturated solution of levofloxacin in 0.1 N HCl was titrated withNaOH. After each addition of the base, the solution was shaken byvortexing. An aliquot of the sample solution was removed, centrifugedand the supernatant analyzed by UV spectroscopy at 288 nm. The samesolution was back titrated with HCl.

Results

The pH-solubility profile of levofloxacin is shown in FIG. 16. Bytitrimetery levofloxacin exhibited a solubility of 25.4 mg/ml at pH 7.3.However contrary to the results of the shaking experiments, thesolubility by titrimetry decreased below pH 6.5 which can be attributedto the common ion effect. Since a solution of levofloxacin was preparedin HCl, a hydrochloride salt of levofloxacin would have formed insolution. Further addition of chloride ions in the form of hydrochloricacid would suppress the solubility of the hydrochloride salt.

pKa Determination

By Titrimetery

Experimental Methodology

A solution of levofloxacin (18 mg/g) was prepared in water (18.45 mg/g).The initial pH of the solution was 7.36. This solution was titrated with1 N HCl. Measured aliquots of HCl were added and the pH recorded aftereach addition. Titration was continued till a pH of 1.

In order to determine the acidic pKa, a solution of levofloxacin (18.38mg/g) was prepared in 0.1 N HCl. The initial pH of the solution was1.32. The solution was titrated with I N NaOH. Titration was continuedtill a pH of 6.55.

Results

FIG. 17 shows a plot of pH Vs volume of titrant added for the titrationof levofloxacin with HCL. This data was fit into the following equation:V_(t) [OH]=K_(b). V_(ep) K_(b). V_(t), where V_(t)=Volume of titrantadded; V_(ep) volume of titrant added till the equivalance point;[OH⁻]=hydroxide ion concentration=Kw/[H⁺]; [H⁺]=hydronium ionconcentration=10^(−pH).

A plot of V_(t) [OH⁻] Vs V_(t) gave a straight line (FIG. 18). Datashown is from the pre-equivalence point region. From the slope we get:Slope: K_(b)=2.09×10⁻⁸; pK_(b)=−log K_(b)=7.7; pK_(a) 14−pK_(b)=6.3.

FIG. 19 shows a plot of pH Vs volume of titrant added for the titrationof levofloxacin with NaOH. Acidic pKa was difficult to calculate becauseit was quite low (<2.0). However, a rough approximation of pKa can bemade as the pH at half the equivalence point. From the plot dpH/dV vsvolume of titrant (V_(t)) (FIG. 20), the equivalence point is at 250 IA.The pH at half the equivalence point (i.e. when V_(t)=125 μl) is 1.6. Sothe acidic pKa ˜1.6.

By UV Spectroscopy

Experimental Methodology

Dilute solutions of levofloxacin (0.013 mg/ml) were prepared in severalbuffers. The buffers used were HCL (pH 1,2), acetate (pH 4,5), phosphate(pH 6,7,8) and borate (9,10). The levofloxacin solutions were analyzedby UV spectroscopy at 257 nm.

Results

A plot of pH Vs Absorbance of the levofloxacin solution at 257 nm isshown as FIG. 21. This data was fitted into a modified HendersonHasselbach equation: Abs_(observed)=Abs_(HA)[H⁺]+Abs_(A).[H⁺]Ka+[H⁺]Ka+[H⁺], where Abs_(observed)=Absorbance of levofloxacinsolution; Abs_(HA)=Absorbance of levofloxacin solution pH 1.2;Abs_(A-)=Absorbance of levofloxacin solution at pH 7.8; [H⁺]=hydroniumion concentration=10⁻pH. The fitted equation provided an estimate ofpKa=5.91.

Example 9 Levofloxacin Salt Formation

The goal of this study was to prepare various salt forms of levofloxacinthat may gain AUC shape-enhancement properties through decreasedsolubility and/or dissolution. These benefits may alter thepharmacodynamic properties of levofloxacin following pulmonaryadministration using nanoparticle suspension or dry powder inhalation.These formulations may be optimized to prolong the release oflevofloxacin from decreased solubility salt forms. These properties mayalso be incorporated into other fluoroquinolone antibiotics including,without limitation gemifloxacin, gatifloxacin, norfloxacin,tosufloxacin, sitafloxacin sarafloxacin, prulifloxacin, andpazufloxacin. Studies are underway to characterize various salt formsand co-preciptates of gemifloxacin for taste masking, AUCshape-enhancement, nanoparticle suspension and dry powder inhalationadministration. Other approaches currently being investigated includespray-dry and in situ micronization techniques.

For suspension and powder formulations, a specific salt form can provideimportant physical and chemical characteristics that may have impacts onthe product performance. For a AUC shape-enhancing formulation, theobjective of salt selection was to decrease the solubility and/or reducethe dissolution rate of levofloxacin. The acid counterions can beselected by:

Manipulation of melting point: An increase in melting point is usuallyaccompanied by a reduction in salt solubility. Salts formed from planar,high melting aromatic acids generally yield crystalline salts of highmelting point.

Manipulation of hydrophobicity: Salts formed with hydrophobic conjugateacids are hydrophobic and difficult to wet, and may ultimately lead toprolonged dissolution.

Examples of acids have been selected for salt preparation are listed asfollows: a) Pamoic acid (embonic acid); b) 2-naphthalene sulfonic acid(napsylic acid); c) Oleic acid; d) Xinafoic acid; e) Stearic acid; f)Lauryl Sulfonate (estolate).

Other factors considered include salt surface properties, polymorph andchemical stability.

Description

The objective of the study was to prepare salt forms of levofloxacin inorder to reduce its solubility and/or dissolution rate. The goal was to:(a) make levofloxacin less soluble through salt formation with suitableexcipient(s); (b) prepare salt forms of levofloxacin that will have alower solubility and/or dissolution rate than the free base. Toaccomplish these tasks, efforts were concentrated on preparing salts atthe basic site of the molecule (pKa ˜6.8).

Pamoic acid (mp=280° C.) and napsylic acid (mp=125° C.) possess planar,hydrophobic structures that were expected to provide hydrophobiccharacter to the salt. The high melting point of pamoic acid may yield ahigh melting crystalline salt form. Oleic acid was chosen primarilybecause it is approved for lung delivery. It has a low melting point (4°C.) which may not satisfy the condition under (1), however it was hopedthat the long aliphatic chain may impart sufficient hydrophobicity todecrease solubility. Xinafoic acid (mp=195° C.) was also selected forsalt formation as it also possess planar, hydrophobic structures that isexpected to provide hydrophobic character to the salt. The rationale forchoosing stearic acid and lauryl sulfonate (estolate) was similar tooleic acid, only their lung toxicities are unknown. Estolate is approvedfor oral delivery (erythromycin estolate has approximately 1/12^(th) thesolubility of the free base, and is formulated as an oral suspension).

Salt Formation

In general, levofloxacin base and the acid were dissolved in a suitablevolatile, organic solvent (1:1 molar ratio), and stirred at roomtemperature. Any crystallized product formed was filtered, dried, andcharacterized. Characterization would consist of DSC, FTIR, andelemental analysis.

Formation and Characterization of Co-Crystals of Levofloxacin withPamoic Acid

Experimental Methodology

Formation of Co-Crystals of Levofloxacin with Pamoic Acid

0.31 g (0.8 mM) of pamoic acid was dissolved by stirring in 100 ml oftetrahydrofuran (THF). To this, 0.30 g (0.8 mM) of levofloxacin wasadded, dissolved by stirring and the resulting solution refluxed for 2.5hrs. The suspension formed was cooled to room temperature, filtered andthe precipitate obtained was dried in vacuum at about 70° C. for 3hours.

Characterization

Thermal analysis. Thermal analysis of (a) pamoic acid (b) levofloxacin(c) levofloxacin pamoate co-crystallized precipitate (d) physicalmixture of pamoic acid and levofloxacin was performed using aDifferential Scanning calorimeter (TA Instrument DSC Q1000). 2-5 mg ofeach sample was weighed into pan, sealed and heated at 10° C./min from25° C. to 300° C. under nitrogen.

Fourier transform Infrared (FT-IR) spectroscopy. FT-IR spectroscopy of(a) pamoic acid (b) levofloxacin (c) levofloxacin pamoateco-crystallized precipitate (d) physical mixture of pamoic acid andlevofloxacin was carried out using a FTIR spectrometer (ModelIRPrestige-21, Shimadzu).

Saturation solubility. Saturation solubility of levofloxacin andlevofloxacin pamoic acid co-crystallized precipitate was determined byequilibrating excess amount of solid with water. The suspensions wereadjusted to pH's 4, 5, 6 and 7 with HCL, shaken, centrifuged and thesupernatant analyzed by UV spectroscopy at 288 nm.

Results

Thermal analysis. DSC scans of (a) pamoic acid (b) levofloxacin (c)levofloxacin pamoate co-crystal precipitate (d) physical mixture ofpamoic acid and levofloxacin are shown as FIGS. 22A, 22B, 22C, and 22D.Pamoic acid and levofloxacin show sharp endotherms at 330° C. and 239°C., respectively, which most likely would be due to the melting ofpamoic acid and levofloxacin, respectively. The DSC profile oflevofloxacin pamoate co-crystals showed one major endotherm at 210° C.,while a 1:1 molar admixture of levofloxacin and pamoic acid displayedbroad endotherms at 129° C. and 220° C.

FTIR. FTIR spectra obtained from (a) pamoic acid (b) levofloxacin (c)levofloxacin pamoate co-crystallized precipitate (d) physical mixture ofpamoic acid and levofloxacin are displayed as FIGS. 23A, 23B, 23C, and23D. The high intensity absorption bands at 1650 cm⁻¹ in the FTIRspectra of pamoic acid, which is due to the stretching of C=0 group isgreatly reduced in the co crystal.

Saturation solubility. Table 31 displays the saturation solubility dataof levofloxacin and levofloxacin pamoate at different pH's. Solubilitywas determined in water, since buffer acids have an effect on thesolubility of levofloxacin. However after shaking levofloxacin or saltsolutions in water, the pH shifted, especially the levofloxacin solutionat pH 5 shifted to pH 1.6. As the solution at pH 5 is between the twopKa's of levofloxacin (˜1.6 and ˜6), such a solution will have a lowerbuffer capacity and hence the pH shift. Solutions at pH's near the pKa'sof the drug have a high buffer capacity and resist pH changes.Solubility of levofloxacin pamoate was considerably less than that oflevofloxacin at all pH's.

TABLE 31 Saturation Solubility Data of Levofloxacin and LevofloxacinPamoate. Levofloxacin pH before shaking 3.97 5.06 5.96 pH after shaking4.21 1.63 5.70 Solubility (mg/ml) 158.28 225.04 297.20 Levofloxacin pHbefore shaking 4.06 4.98 5.98 7.00 pamoate pH after shaking 5.63 6.306.66 7.32 Solubility (mg/ml) 0.34 0.29 0.24 0.44

Interpretation

Since the co-crystallized precipitate of levofloxacin pamoate has adifferent melting point and FTIR spectra from that of levofloxacin,pamoic acid or their physical mixture, it is possible that the equimolarcomplex of the levofloxacin with pamoic acid might be the saltlevofloxacin pamoate, having a considerably less solubility thanlevofloxacin.

Formation and Characterization of Co-Crystals of Levofloxacin withXinafoic Acid

Experimental Methodology

Formation of of Co-Crystals of Levofloxacin with Xinafoic Acid

1.004 g (2.7 mM) of levofloxacin was dissolved by refluxing in 80 mlethyl acetate. To this, 0.51 g (2.7 mM) of xinafoic acid dissolved in 35ml of ethyl acetate was added and the solution cooled overnight understirring conditions to ambient temperature. The suspension obtained wasfiltered, and the precipitate dried under vacuum at 75° C. for about 3.5hours.

Characterization

Thermal analysis. Thermal analysis of (a) xinafoic acid (b) levofloxacinxinafoate co-crystallized precipitate was performed using a DifferentialScanning calorimeter (TA Instrument DSC Q1000). 2-5 mg of each samplewas weighed into pan, sealed and heated at 10° C./min from 25° C. to300° C. under nitrogen.

Fourier transform Infrared (FT-IR) spectroscopy. FT-IR spectroscopy of(a) xinafoic acid (b) levofloxacin xinafoate co-crystallized precipitatewas carried out using a FTIR spectrometer (Model IRPrestige-21,Shimadzu).

Saturation solubility. Saturation solubility of levofloxacin xinafoicacid co-crystallized precipitate was determined by equilibrating excessamount of solid with water. The suspensions were adjusted to pH's 4, 5,6 and 7 with HCL, shaken, centrifuged and the supernatant analyzed by UVspectroscopy at 288 nm.

Results

Thermal analysis. DSC profiles of (a) xinafoic acid (b) levofloxacinxinafoate co-crystallized precipitate are shown as FIGS. 24A and 24B.Levofloxacin xinafoate co-crystallized precipitate exhibits a meltingendotherm at 196° C., which is different than that of xinafoic acid(216° C.) and levofloxacin (239° C.).

FTIR. FTIR spectra obtained from (a) xinafoic acid (b) levofloxacinxinafoate co-crystallized precipitate are displayed as FIGS. 25A and25B. The FTIR spectrum of the co crystal exhibit transmittance minima'sat wave numbers different from that of xinafoic acid and levofloxacin.

Saturation solubility. Table 32 shows the saturation solubility data oflevofloxacin xinafoate at different pH's. Solubility of the xinafoatesalt was intermediate between that of the levofloxacin base andlevofloxacin pamoate co crystal.

TABLE 32 Saturation Solubility Data of Levofloxacin Xinafoate Co-CrystalLevofloxacin pH before shaking 4.05 4.94 6.00 6.95 xinafoate pH aftershaking 4.78 5.37 6.30 7.17 Solubility (mg/ml) 21.69 12.95 4.59 5.95

Interpretation

The co-crystallized precipitate of levofloxacin pamoate has a differentmelting point and FTIR spectra than that of levofloxacin and xinafoicacid, suggesting a possible formation of a levofloxacin xinafoate salt.This salt has intermediate solubility between levofloxacin andlevofloxacin pamoate.

Formation and Characterization of Co-Crystals of Levofloxacin withStearic Acid

Experimental Methodology

Formation of Co-Crystals of Levofloxacin with Stearic Acid

0.77 g (2.07 mM) of stearic acid was dissolved by heating and sonicationin 40 ml of methanol. To this, 1.00 g (2.07 mM) of levofloxacindissolved in 60 ml of methanol was added. The resulting solution washeated at 55° C. for about 15 minutes, followed by cooling to roomtemperature and then at −20° C. The suspension obtained was filtered.

Characterization

Thermal analysis. Thermal analysis of (a) stearic acid (b) levofloxacinstearate co-crystallized precipitate (c) physical mixture of stearicacid and levofloxacin was performed using a Differential Scanningcalorimeter (TA Instrument DSC Q1000). 2-5 mg of each sample was weighedinto pan, sealed and heated at 10° C./min from 25° C. to 250° C. undernitrogen.

Fourier transform Infrared (FT-IR) spectroscopy, FT-IR spectroscopy of(a) stearic acid (b) levofloxacin stearic acid co-crystallizedprecipitate (d) physical mixture of stearic acid and levofloxacin wascarried out using a FTIR spectrometer (Model IRPrestige-21, Shimadzu).

Saturation solubility. Saturation solubility of levofloxacin andlevofloxacin stearic acid co-crystallized precipitate was determined byequilibrating excess amount of solid with water. The suspensions wereadjusted to pH's 4, 5, 6 and 7 with HCL, shaken, centrifuged and thesupernatant analyzed by UV spectroscopy at 288 nm.

Results

Thermal analysis. DSC scans of (a) stearic acid (b) levofloxacinstearate co-crystal precipitate (d) physical mixture of stearic acid andlevofloxacin are shown as FIGS. 26A, 26B and 26C. Stearic acid andlevofloxacin show sharp endotherms at 76.4° C. and 239° C.,respectively, which most likely would be due to the melting of stearicacid and levofloxacin, respectively. The DSC profile of levofloxacinstearic acid co-crystals showed two sharp endotherms at 88.03° C. and138.54° C. and minor endotherms at 231° C. and 242.72° C. The minorendotherms might be due to melting of trace quantities of residuallevofloxacin in the original sample. A 1:1 molar admixture oflevofloxacin and stearic acid displayed endotherms at 68.87° C., 134.43°C. and 240.74° C. and minor endotherms at 79.73° C. and 86.74° C.

FTIR. FTIR spectra obtained from (a) stearic acid (b) levofloxacinstearic acid co-crystallized precipitate (c) physical mixture of stearicacid and levofloxacin are displayed as FIGS. 27A, 27B, and 27C. The C=0stretch band is seen at 1700, 1705 and 1721 cm⁻¹ in stearic acid,co-crystallized precipitate and physical mixture, respectively.

Saturation solubility. Table 33 shows the saturation solubility data oflevofloxacin stearic acid co-crystals at different pH's.

TABLE 33 Saturation Solubility Data of Levofloxacin Stearic AcidCo-Crystals Levofloxacin pH before shaking 4.05 5.02 6.01 6.96 stearatepH after shaking 3.36 5.05 6.02 6.98 Solubility(mg/ml) 0.86 1.30 2.201.64

Interpretation

The DSC profile of levofloxacin stearic acid co-crystal precipitateshows two endotherms. One of these endotherms might be due to melting ofthe cocrystals. The nature of the second endotherm has to beinvestigated. Since the levofloxacin stearic acid co-crystal precipitatehas a solubility values lower than of levofloxacin, it is possible thatthe precipitate might be the salt, levofloxacin stearate salt.

Formation and Characterization of Co-Crystals of Levofloxacin with OleicAcid

Experimental Methodology

Formation of Co-Crystals of Levofloxacin with Oleic Acid

0.78 g (2.76 mM) of oleic acid was dissolved in 10 ml chloroform. Tothis, 1.025 (2.76 mM) of levofloxacin dissolved in 10 ml of chloroformwas added. The resulting solution was mixed thoroughly and evaporated at40° C.

Characterization

Thermal analysis. Thermal analysis of (a) oleic acid (b) levofloxacinoleate co-crystal precipitate (c) physical mixture of oleic acid andlevofloxacin (50:50) (d) physical mixture of oleic acid and levofloxacin(10:90) and (e) physical mixture of oleic acid and levofloxacin (90:10)was performed using a Differential Scanning calorimeter (TA InstrumentDSC Q1000). 2-5 mg of each sample was weighed into pan, sealed andheated at 1° C./min or 10° C./min from 25° C. to 250° C. under nitrogen.

Fourier transform Infrared (FT-IR) spectroscopy. FT-IR spectroscopy of(a) oleic acid (b) levofloxacin oleic acid co-crystallized precipitate(d) physical mixture of oleic acid and levofloxacin was carried outusing a FTIR spectrometer (Model IRPrestige-21, Shimadzu).

Kinetic solubility determination. Levofloxacin oleate co-crystallizedprecipitate (50 mg) was suspended in 2 ml of water. The suspension wasadjusted to pH 7 with HCL and shaken. The solubility of theseco-crystals was determined at various time intervals. This study wasperformed at room temperature and at 40° C. The kinetic solubility of anequimolar physical mixture of levofloxacin and oleic acid was alsoperformed and compared to that of the co-crystals at 40° C.

Results

Thermal analysis. DSC scans of (a) oleic acid (b) levofloxacin oleateco-crystal precipitate (c) physical mixture of oleic acid andlevofloxacin (50:50) (d) physical mixture of oleic acid and levofloxacin(10:90) and (e) physical mixture of oleic acid and levofloxacin (90:10)are shown as FIGS. 28A, 28B, 28C, 28D, and 28E. Oleic acid thermogramshows endotherms at −6.15° C. and 13.05° C. The endotherm at −6.15° C.corresponds to gamma-alpha oleic acid phase transition (Crowley K. J,1999). Levofloxacin oleate co-crystallized precipitate shows endothermat 127.69° C., while an equimolar physical mixture of levofloxacin witholeic acid shows endotherms at 123.69° C., 179.35° C. and 224° C. Theequimolar physical mixture is showing an endotherm which is close tomelting point of the co-crystals, suggesting a possible reaction betweenoleic acid and levofloxacin in the solid state. To investigate thisphenomenon, DSC on physical mixtures of levofloxacin and oleic acid(90:10) and (10:90) were performed. Physical mixture of levofloxacin andoleic acid (10:90) shows major endotherm at 10.33° C. (possible meltingof oleic acid) and at 281° C. It does not show endotherm near themelting point of co-crystals. A levofloxacin oleic acid (90:10) physicalmixture shows no melting endotherm at 10° C. for oleic acid. It exhibitsendotherms at 79.77° C. and at 128° C. (close to the melting point ofthe co-crystals), suggesting a possible reaction of levofloxacin andoleic in presence of high amounts of levofloxacin.

FTIR.

The FTIR spectra of oleic acid shows a C=0 stretch intense peak at 1710cm-1 and O—H in-plane and out-of-plane bands at 1462 and 937 cm-1,respectively.

The high intensity absorption bands at 1710 cm⁻¹ in the FTIR spectra ofoleic acid, which is due to the stretching of C=0 group is slightlyreduced in the co crystal. The O—H in plane and out of plane bands at1462 and 937 cm-1 in the oleic acid are absent in the co-crystal. Alsothe FTIR spectrum of the physical mixture is different from that of thesalt (FIGS. 29A, 29B, and 29C).

Kinetic Solubility Determinations

FIG. 30 displays the data for the kinetic solubility experimentsperformed with the co-crystallized precipitate at room temperature andat 40° C. The solubility of the co-crystals at room temperature is about0.9 mg/ml, which remains constant throughout the period of the study. At40° C., the solubility increased from 1.17 mg/ml at 15 minutes to 1.86mg/ml at 4 hrs which remained almost constant till 24 hrs. Thesolubility profile of the equimolar physical mixture at 40° C. looksdifferent from that of the co-crystals. The physical mixture has ahigher solubility (9.16 mg/ml at 24 hrs) as compared to the co-crystals(1.89 mg/ml at 24 hrs).

Interpretation

The DSC data of the equimolar physical mixture exhibits an endothermnear the melting endotherm of the co-crystallized precipitate. Howeverthe FTIR and solubility data of the co-crystals is different than thatof physical mixture, with the co-crystals having a low saturationsolubility. The saturation solubility of the co-crystals is 0.9 mg/mltemperature as opposed to 25 mg/ml for levofloxacin base.

However the levofloxacin oleate salt is waxy in nature which might bedifficult to grind/micronize and thereby formulate. It is reported thatthe tacky and deformable properties of a wax-like drug fatty acid salt,propranolol oleate made particle size reduction difficult (Crowley. J.,et al, International journal of Pharmaceutics, 2000, 211 (1-2): 9-17.

Dissolution Rate Studies

Levofloxacin Xinafoate

Experimental Methodology

50 mg of the levofloxacin xinafoate salt was suspended in a dissolutionbath containing 500 ml of pH 7.4 Tris buffer at 37° C. and rotated bymeans of paddles at 100 rpm. 5 ml samples were removed at periodic timeintervals and replaced with same volume of plain buffer.

Results

The dissolution profile of levofloxacin xinafoate is shown as FIG. 31.It is seen that the dissolution rate of levofloxacin xinafoate in theearlier stages of 2-10 minutes is faster than that seen from 10-30minutes. When levofloxacin xinafoate is added to the dissolution media,it gets dispersed as a fine powder, and the dissolution from these fineparticles is faster, approximately 1.24 mg/min (FIG. 32). With time, thepowder gets agglomerated and moves in a vortex created by the paddle,thereby decreasing the dissolution rate to 0.28 mg/min (FIG. 33).

Levofloxacin

Experimental Methodology

200 mg of levofloxacin was suspended in a dissolution bath containing500 ml of pH 7.4 Tris buffer at 37° C. and rotated by means of paddlesat 100 rpm. 5 ml samples were removed at periodic time intervals andreplaced with same volume of plain buffer.

Results

The dissolution profile of levofloxacin is shown as FIG. 34. Aslevofloxacin has a higher solubility than the salts its dissolution rateis very fast. With levofloxacin too, the earlier dissolution was fromfinely dispersed particles and hence a faster dissolution rate. In thelater stages the particles agglomerated and reduced its dissolutionrate.

Levofloxacin Pamoate

Experimental Methodology

10 mg of the levofloxacin pamoate salt was suspended in a dissolutionbath containing 500 ml of pH 7.4 Tris buffer at 37° C. and rotated bymeans of paddles at 100 rpm. 5 ml samples were removed at periodic timeintervals (2, 5, 10, 15, 20, 25, 30, 45, 60, 120, 240, 1320 and 1440minutes) and replaced with same volume of plain buffer. The study wasperformed in duplicate.

Results

The dissolution profile of levofloxacin pamoate is shown as FIG. 35. Itis seen that the dissolution rate of levofloxacin pamoate in the earlierstages of 2-10 minutes is faster than that seen from 10-60 minutes. Whenlevofloxacin pamoate, is added to the dissolution media, it getsdispersed as a fine powder, and the dissolution from these fineparticles is faster, approximately 0.146 mg/min (FIG. 36). With time,the powder gets agglomerated and moves in a vortex created by thepaddle, thereby decreasing the dissolution rate to 0.0331 mg/min (FIG.37).

Levofloxacin Stearate

Experimental Methodology

25 mg of the levofloxacin stearate salt was suspended in a dissolutionbath containing 500 ml of pH 7.4 Tris buffer at 37° C. and rotated bymeans of paddles at 100 rpm. 5 ml samples were removed at periodic timeintervals (2, 5, 10, 15, 20, 25, 30, 45, 60, 120, 240, 1320 and 1440minutes) and replaced with same volume of plain buffer.

Results

The dissolution profile of levofloxacin stearate is shown as FIG. 38. Itis seen that the dissolution rate of levofloxacin stearate in theearlier stages of 2-10 minutes is 0.499 mg/min (FIG. 39) which is fasterthan that seen from 10-30 minutes (0.161 mg/min) (FIG. 40).

Dissolution of levofloxacin and salts was carried out with quantitiessuch that the concentration of the dissolved solute in the dissolutionbath never reached more than 10% of its saturation solubility. This wasdone in an attempt to maintain sink conditions.

Pending the dissolution rate of these and other salt forms andco-precipitates, these AUC shape-enhancement forms of levofloxacin,gemifloxacin and other fluoroquinolone antibiotics these forms may bebest suited for nanoparticle suspension (solubilities <100 ug/ml, slowdissolution rates) or micron-size dry powders (solubilities >100 ug/ml,quicker dissolution rate than that best for nanosuspension. Thenanoparticle suspensions may be administered by nebulization using jet,ultrasonic, or vibrating mesh technologies, while dry powderformulations may be administered using either active or passive drypowder inhalers.

Example 11 Levofloxacin Solid Lipid Nanoparticles

The goal of this study was to prepare solid lipid nanoparticles oflevofloxacin to obtain AUC shape-enhancing properties through decreasedsolubility and dissolution. These benefits may enhance thepharmacodynamic properties of levofloxacin following pulmonaryadministration using nanoparticle suspension or dry powder inhalationformulations. These formulations are being optimized to prolong therelease of levofloxacin from decreased solubility salt forms. Theseproperties may also be incorporated into other fluoroquinoloneantibiotics including, without limitation gemifloxacin, gatifloxacin,norfloxacin, tosufloxacin, sitafloxacin sarafloxacin, prulifloxacin, andpazufloxacin. Studies are also underway to characterize various lipidnanoparticle forms of gemifloxacin for taste masking, AUCshape-enhancement, nanoparticle suspension and dry powder inhalationadministration. Other approaches for solid lipid nanoparticle currentlybeing investigated include spray-dry and in situ micronizationtechniques.

Preformulation Studies

Partitioning of each compound (including levofloxacin salts and metalcation complexes) into 1-octanol were determined at different relevantpH values. Partitioning as a function of time may also be evaluated todetermine whether dissociation of levofloxacin occurs (from both saltsand complexes), and in the case of salts, also to determine if selectivepartitioning of the fatty acid component occurs over time. Compound(s)with significant partitioning (log P>2.0) was evaluated for theirsolubility in various lipid melts. Additionally, partitioning of alipophilic fluoroquinolne (if available) is also being studied, and itssolubility in different lipid melts will be evaluated. A lipid in whichdrug is sufficiently soluble will be selected for formulation of solidlipid nanoparticles. A prerequisite to obtain a sufficient loadingcapacity of the drug in solid lipid nanoparticles was a high solubilityof drug in the lipid melt.

Formulation of Solid Lipid Nanoparticles

Formulation of solid lipid nanoparticles typically involves dissolvingthe drug in a lipid melt, followed by dispersion of the drug-containingmelt in a hot aqueous surfactant solution. The coarse dispersion ishomogenized using a Microfluidizer® to obtain a nanoemulsion. Coolingthe nanoemulsion to room temperature will re-solidify the lipid, leadingto formation of solid lipid nanoparticles. Optimization of formulationparameters (type of lipid matrix, surfactant concentration andproduction parameters) will be performed so as to achieve a prolongeddrug delivery.

Characterization of Solid Lipid Nanoparticles

The nanoparticles are being characterized for size and zeta potentialusing a Dynamic Light Scattering instrument while Laser diffraction willbe used for the detection of large microparticles.

Upun completion of the synthesis, differential scanning calorimetricstudies will be performed to investigate any possible modificationsinduced in the physical form of the lipid.

In vitro drug release testing will be done using appropriatemethodology.”

Example 12 Levofloxacin Metal Ion Complexes

The goal of this study was to prepare levofloxacin of various chelatesalt forms to obtain gain taste-masking properties, AUC shape-enhancingproperties through changes in solubility, dissolution and/orbioavailability. These benefits may enhance the pharmacodynamicproperties of levofloxacin following pulmonary administration usingnanoparticle suspension, dry powder inhalation or simple liquidformulations. These formulations may be optimized to create AUCshape-enhancing formulations of levofloxacin from altered solubility, orslow-release or low bioavailability chelates. These properties may alsobe incorporated into other fluoroquinolone antibiotics including,without limitation gemifloxacin, gatifloxacin, norfloxacin,tosufloxacin, sitafloxacin sarafloxacin, prulifloxacin, andpazufloxacin. Studies are also underway to characterize various andchelate forms of gemifloxacin for taste masking, AUC shape-enhancement,nanoparticle suspension and dry powder inhalation administration.

Preparation of Levofloxacin-Metal Ion Complexes

Preliminary Studies

A mixture of levofloxacin and a salt of a given cation was solubilizedin deionized water and titrated with sodium hydroxide. The titrationcurve was compared against one obtained for levofloxacin alone to assessformation of levofloxacin-metal complex as described in PhysicalPharmacy (4th Edition) by Alfred Martin (pp 261-263). Salts of variousmetal cations (e.g. Ca2+, Mg2+, etc) were then evaluated to identifysuitable candidate(s) for subsequent evaluations. Different molar ratiosof cations and levofloxacin were also evaluated.

Preparation of Complexes

Levofloxacin solutions were titrated against aqueous solutions ofselected metal salts. Titrations were carried out at a constant pH.Formation of complexes were monitored by different methods includingtitrimetry, spectrofluorometry, solubility, etc. as applicable. The endpoint of the complexation reaction depended on the method adopted.

Characterization of Levofloxacin Complexes

Levofloxacin-metal cation complexes were characterized forstoichiometry, formation constants and dissociation kinetics usingappropriate methodology.

Goals

To formulate and characterize levofloxacin complexes with metal cations(di- and tri-valent).

Assessment of Complexation

Preliminary investigations suggested that levofloxacin forms solublecomplexes with metal cations. As a result, evaluation of thecomplexation process by precipitation was not possible. Other approachesthat were attempted are described below.

Titrimetry

This approach was based on the assumption that the carboxylic acidmoiety of levofloxacin is involved in complex formation with a givenmetal cation and that complexation results in the release of a protonfrom levofloxacin. The concentration of released protons would thus beproportional to the extent of complexation (depending on the bindingconstant) and the stoichiometry of the complex (Physical Pharmacy:4^(th) Edition by Alfred Martin; pp-261-263).

Experimental Methodology

About 0.35 mmoles of levofloxacin (in 16 mL of deionized water) weretitrated with 6N NaOH in the presence and absence of salt of a metalcation (equimolar). Levofloxacin solutions were acidified to pH valuesless than 2.0 with 6N HCl prior to titration with NaOH. Salts of metalcations used include calcium chloride, magnesium chloride, ferrouschloride, zinc chloride, aluminum sulfate and aluminum chloride.

Results

As shown in FIG. 41, titrations performed in the presence of metalcations resulted in a positive shift of the titration curves as comparedto the one obtained with levofloxacin alone suggesting that additionalNaOH (titrant) is required to obtain a specific pH of the solution inthe presence of metal cation. The magnitude of the shift in titrationcurve at any point would represent moles of proton released due tocomplexation and hence moles of complexed levofloxacin.

Extent of complexation (binding and/or stoichiometry) appears toincrease in the order Ca⁺<Mg²⁺<Zn²⁺=Fe²⁺<Al³⁺, which is in reasonableagreement with existing literature.

Note: It was noted from the literature that aluminum chloride andaluminum sulfate have acid-like properties and would lower the pH ofaqueous solutions. Consequently, the titration curves obtained withA1C13 and Al2(504)3 may not provide conclusive information oncomplexation with levofloxacin.

Dual Titration

In this approach levofloxacin solution was titrated with a solution of agiven metal cation to observe a drop in pH presumably due to release ofprotons through complexation. This was followed by addition of NaOH torevert back to the initial pH of the levofloxacin solution (prior toaddition of solution of cation). This enables determination of thefraction of levofloxacin in the complexed form at a given pH.

Experimental Methodology

About 1.55-1.72 mmoles of levofloxacin were solubilized in deionizedwater and the resulting solution was acidified with 6N HCl to thedesired initial pH. This acidified levofloxacin solution was titratedwith a known volume of concentrated solution of a given metal cation(Ca²⁺, Mg²⁺, Fe2⁺ and Zn²⁺). The change in pH was neutralized (to theinitial pH) by the addition of 6N NaOH and volume of NaOH solution addedwas recorded. Addition of solution of metal cation followed byneutralization with NaOH was continued until further addition ofsolution of metal cation failed to result in pH change of thelevofloxacin solution, which would indicate endpoint of complexation.The cumulative amounts of metal cation added were plotted againstcumulative amounts of NaOH required to neutralize the change in pH(FIGS. 42-45).

Results

From FIGS. 42-45, the plateau regions were extrapolated to obtain totalamount of NaOH required to neutralize the change in pH due tocomplexation. These values also represent the amounts of levofloxacin inthe complexed form (assuming that complexation of levofloxacin resultsin an equimolar release of protons). Amounts of levofloxacin in thecomplexed form with Ca²⁺, Mg²⁺, Fe²⁺ and Zn²⁺ are 0.8, 1.0, 1.3 and 1.1mmoles, respectively. These represent 46.5, 64.5, 77.8 and 64.5%complexation for Ca²⁺, mg²⁺, Fe²⁺ and Zn²⁺, respectively. It should benoted that % complexation would depend on the total concentrations oflevofloxacin.

The binding constants as well as the stoichiometry of complexation forthe levofloxacin complexes with the metal cations were determined asfollows: M+nA 4

MA_(n)K_(b), where M, A and MA_(n) represent the metal cation,levofloxacin and the complex, respectively. K_(b) would be theequilibrium binding constant. The above reaction assumes that ‘n’ molesof levofloxacin react with one mole of metal to yield one mole ofcomplex.

Equation 1 is K_(b)=[MA_(n)]/{[M][A]^(n)} (units M^(−n)), where [MA_(n)]is the concentration of complex formed; [M] and [A] are theconcentrations of the unbound metal and unbound levofloxacin,respectively.

Rearranging Eq.1; Equation 2 is [MA_(n)]/[A]^(n)=K_(b)*[M], where[A]=[A]_(Total)−[A]_(bound)=[A]_(Total) [NaOH]_(used); [M]=[M]_(Total)[M]_(bound)=[M]_(Total)[Na0H]_(used)/n; and[MA_(n)]=[A]_(bound)/n=[NaOH]/_(used)/n.

Note: [NaOH]_(used) the concentration of sodium hydroxide used at anygiven point to neutralize the change in pH caused by the addition ofmetal cation (presumably due to complexation).

Equation 2 can be modified to obtain Equation 3, which is:[A]_(bound)[A]_(n)=nK_(b)*[M]. It is inferred from Equation 3 that aplot of [M] versus [A]_(bound)[A]_(n) would result in a straight linewith a slope of nK_(b) when, n=1, for a 1:1 complex; n=2, for a 2:1complex; n=3, for a 3:1 complex etc.

Shown below in FIGS. 46A-46B, 47A-47B, 48A-48B, and 49A-49B are theseplots for Ca²⁺, Mg²⁺, Fe²⁺ and Zn²⁺, respectively.

As shown in FIGS. 46A-46B, 47A-47B, 48A-48B, and 49A-49B, for each ofthe cations evaluated a plot of [A]bound/[A]n versus nK_(b)*[M] waslinear when n=2 (for Ca²⁺ n=2 resulted in a better fit than n=1). Theseresults suggest that levofloxacin complexes with Ca²⁺, Mg²⁺, Fe²⁺ andZn²⁺ are formed with a stoichiometry of 2 moles of drug per mole ofcation (2:1).

Using n=2, the binding constants for the above complexes can bedetermined from the slopes of the respective linear plots.

The binding constants for 2:1 complexes represented as log (K_(b)) areas follows: Ca²⁺=2.75, Mg²⁺=3.69, Zn²⁺=4.44, Fe²⁺=4.54.

Solubility

This method allows for a relatively simple way of determining thestoichiometry of complexation. The approach involved evaluation ofsolubility of the drug (levofloxacin) in the presence of increasingconcentrations of complexation agent (a given metal cation). The totalsolubility of the drug (complexed+uncomplexed) was expected to increaselinearly owing to complexation and to reach a plateau corresponding tothe saturation solubility of both the drug and the complex.Determination of the stoichiometry from such a solubility curve wasexplained in detail elsewhere (Physical Pharmacy: 4^(th) Edition byAlfred Martin; pp 265).

Experimental Methodology

Excess quantities of levofloxacin (amounts were recorded) were agitated,in the presence of increasing concentrations of MgCl₂, with 25 mM IVIESbuffer (pH 5.99) using a vortex mixer. The samples were then filteredand the filtrate was diluted appropriately and analyzedspectrophotometrically to determine levofloxacin concentrations (FIG.50).

Results

As shown in FIG. 50, the solubility of levofloxacin did increase withincreasing MgCl2 concentrations. However, beyond the plateau solubility(˜650 mM levofloxacin), further increase in solubility was observed,which is not consistent with the expected profile. This was attributedto the effect of ionic strength on levofloxacin solubility. It isimportant to note that the final pH of all the solutions were constant,albeit greater than 5.99 (final pH ˜7.0).

Subsequently, the experiment was repeated at a constant ionic strengthof ˜1.0M (adjusted with NaCl) and with 0.5M MES buffer (pH 5.99) toenhance the buffer capacity of the solution (FIG. 51).

Spectrofluorometry

This approach was adopted to evaluate levofloxacin complexation based onexisting literature evidence that the complexation process is associatedwith a change in the fluoroquinolone fluorescence properties. Bymonitoring the change in fluorescence emission of levofloxacin in thepresence of different concentrations of a given metal cation it waspossible to determine the binding constant of complexation as well asthe stoichiometry.

Experimental Methodology

The fluorescence emission of levofloxacin was evaluated at excitationand emission wavelengths of 298 nm and 498 nm, respectively. Studieswere conducted at two different pH values i.e. 5.0 (acetate) and 9.0(histidine). A series of solutions containing a constant levofloxacinconcentration but increasing concentrations of a given cation wereanalyzed for fluorescence emission due to levofloxacin. Metal saltsstudied included CaCl₂, MgCl₂, FeCl₂, ZnCl₂ and Al₂(SO₄)₃.

Results

TABLE 34 Fluorescence Characteristics of Levofloxacin in the Presence ofCations Fluorescence of levofloxacin Cation pH 5.0 pH 9.0 ResultsComments Ca²⁺ Change not Change not N/A — significant significant mg²⁺Change not Change not N/A — significant significant Fe²⁺ Decrease in N/AFIG. 3.12 FeCl₂ insoluble emission with (pH 5.0) at pH 9.0 increasingFe²⁺ Zn²⁺ Change not Increase in FIG. 3.13 — significant emission with(pH 9.0) increasing Zn²⁺ Al³⁺ Change not N/A N/A Al₂(SO₄)₃ significantInsoluable at pH 9.0

As shown in Table 34, significant data were obtained only for Fe²⁺ andZn²⁺. For the remaining cations, the relative concentrations oflevofloxacin and the cation need to be further optimized to observe aspecific trend in change in levofloxacin fluorescence.

The influence of increasing concentrations of Fe2+ and Zn2+ onlevofloxacin fluorescence emission are shown in FIGS. 52 and 53,respective.

As described above, both Fe²⁺ and Zn²⁺ appear to form 2:1 complexes withlevofloxacin; however, their influence on levofloxacin fluorescence aredissimilar (FIGS. 52 and 53). The exact reason for this is unclear atthis point.

Samples of Levofloxacin Complexes

Seven samples of levofloxacin complexes were evaluated in vivo forefficacy and pharmacokinetics. Details of the samples tested are shownin Table 35 below.

TABLE 35 Molar Ratios of Levofloxacin Complexes Molar Total Final pHratio Levofloxacin of the Sample identifier Cation used (mg/mL) solutionNB-049-001-06-066A Mg²⁺ 1:1 40.2 6.24 NB-049-001-06-066B Fe²⁺ 1:1 40.16.30 NB-049-001-06-066C Mg²⁺ 1:1 202 5.98 NB-049-001-06-081A Ca²⁺ 1:140.1 6.53 NB-049-001-06-081B Ca²⁺ 1:1 201 6.04 NB-049-001-06-081C Zn²⁺1:1 40 6.33 NB-049-001-06-081D Zn²⁺ 1:1 200 5.69

Conclusions and Next Steps

Results obtained from our dual titration studies suggest thatlevofloxacin forms 2:1 complexes with all the divalent metal cations.The binding constants (log K_(b)) for complexation with Ca²⁺, Mg²⁺, Fe²⁺and Zn²⁺ are 2.75, 3.69, 4.44 and 4.54, respectively.

Example 13 Levofloxacin and Gemifloxacin Formulations with Organic Acids

Experimental Methodology

Levofloxacin solution was prepared by dissolving either 50 or 100 mglevofloxacin base in 15-20 ml water. The initial pH of levofloxacinsolution in water was about 7.3. The pH of the solution was adjustedwith about 10% solution of acid prepared in water. The following acidswere used to adjust the pH of the levofloxacin solution: acetic acid,ascorbic acid, citric, lactic, tartaric and propionic acid. After makingup the volume of the solution to approximately 90% of the final volume,the osmolality of the solution was measured and adjusted to 300 mOsm/kgwith about 20% solution of sodium chloride prepared in water After pHand osmolality adjustment, the volume of the solution was made up toabout 25 ml with water and its surface tension measured. The pH andosmolality were measured after making up the volume and are reported inTable 36. (The exact quantities of levofloxacin weighed, acid requiredto adjust pH, sodium chloride to adjust osmolality and final volume ofsolutions are listed in Table 36). The content of levofloxacin in thesolutions was determined by HPLC.

TABLE 36 Formulations of Levofloxacin with Organic Acids 9.94% Measuredacetic 19.7% Final Vol Final Wt of acid Acetic NaC1 of Levo osmolalitySurface Levo used acid used NaC1 solution conc (mOsm/ tension used (g)(ml) used(g) (ml) used (g) (ml) (mg/ml) kg) Final pH (mN/m) 1.253 1.050.104 0.681 0.134 25.105 49.9 312 6.48 63.2 2.501 2.05 0.204 0.326 0.06425.935 96.4 300 6.53 62.5 9.99% Measured ascorbic 19.7% Final Vol FinalWt of acid ascorbic NaC1 of Levo osmolality Surface Levo used acid usedNaC1 solution conc (mOsm/ tension used (g) (ml) used(g) (ml) used (g)(ml) (mg/ml) kg) Final pH (mN/m) 1.253 3.400 0.339 0.550 0.108 25.13549.8 297 6.40 64.4 2.505 7.400 0.739 0.300 0.059 25.135 99.7 298 6.4762.5 10.05% Measured citric 21.54% Final Vol Final Wt of acid citricNaC1 of Levo osmolality Surface Levo used acid used NaC1 solution conc(mOsm/ tension used (g) (ml) used(g) (ml) used (g) (ml) (mg/ml) kg)Final pH (mN/m) 1.251 1.25 0.126 1.005 0.216 25.12 49.8 299 6.54 61.52.498 2.6 0.261 0.918 0.198 25.82 96.7 301 6.53 61.4 10% Measured lactic21.54% Final Vol Final Wt of acid Lactic NaC1 of Levo osmolality SurfaceLevo used acid used NaC1 solution conc (mOsm/ tension used (g) (ml) used(g) (ml) used (g) (ml) (mg/ml) kg) Final pH (mN/m) 1.258 2.1 0.21 0.7450.160 25.135 50.1 297 6.54 59.4 2.497 4.2 0.42 0.392 0.084 25.605 97.5301 6.63 57.5 10% Measured tartaric 21.54% Final Vol Final Wt of acidTartaric NaC1 of Levo osmolality Surface Levo used acid used NaC1solution conc (mOsm/ tension used (g) (ml) used (g) (ml) used (g) (ml)(mg/ml) kg) Final pH (mN/m) 1.252 1.55 0.155 0.948 0.204 25.180 49.7 2986.51 61.5 Measured 9.79% 21.54% Final Vol Final Wt of propionicpropionic NaC1 of Levo osmolality Surface Levo acid used acid used usedNaC1 solution conc (mOsm/ Final tension used (g) (ml) (g) (ml) used (g)(ml) (mg/ml) kg) pH (mN/m) 1.25281 1.310 0.128 0.737 0.159 25.045  50.02298 6.50 58.1 2.51342 2.610 0.256 0.310 0.067 25.030 100.42 297 6.5752.0

Results

The details about the levofloxacin formulations with organic acids areshown in Table 36. The results of HPLC are shown in Table 37.

TABLE 37 Theoretical and Measured Concentrations of Levofloxacin in theFormulations Theoretical Measured Conc. Concentration Acid (mg/mL)(mg/mi) by HPLC acetic acid 50.05 51.45 acetic acid 99.9 102.32 citricacid 49.91 50.31 citric acid 99.86 102.99 L-ascorbic acid 49.95 50.01L-ascorbic acid 100 102.49 lactic acid 50.05 50.07 lactic acid 97.5495.27 tartaric acid 49.74 51.07

When tartaric acid was used to adjust the pH of the 100 mg/mllevofloxacin solution, a precipitate was formed.

Note: Solutions with acetic acid, citric acid and ascorbic acid wereremade for HPLC analysis and hence the theoretical concentration forthese solutions in Table 36 and Table 37 are different.

Gemofloxacin Formulations with Organic Bases

Experimental Methodology and Results

Gemofloxacin Formulation with Sodium Ascorbate

50.30 mg of Gemifloxacin mesylate (equivalent to 40.37 mg Gemifloxacin)was added to 1.5 ml water. The resulting solution was cloudy. It wasfiltered through a 0.45 micron filter. 1.3 ml of solution was obtainedafter filtering having a pH of 4.28. The pH of this solution wasadjusted to 5.48 with 400 uL of a 10% solution of sodium ascorbateprepared in water (Quantity of base required to adjust pH=0.04 g). Theosmolality of this solution was 308 mOsm/kg, hence sodium chloride wasnot used to adjust osmolality. The final volume of the solution was 1.7ml. Theoretical concentration of gemifloxacin in this formulation wouldbe 20.59 mg/ml. Theoretical concentration=Theoretical quantity ofgemifloxacin in filtered solution (in this case 35 mg Gemifloxacin infiltered 1.3 mL)/Final volume of solution (in this case 1.7 mL).

Gemifloxacin Formulation with Sodium Lactate

50.05 mg of Gemifloxacin mesylate (equivalent to 40.17 mg Gemifloxacin)was added to 1.8 ml water. The resulting solution was cloudy. It wasfiltered through a 0.45 micron filter. 1.52 ml of solution was obtainedafter filtering having a pH of 4.21. The pH of this solution wasadjusted to 5.42 with 180 uL of a 20% solution of sodium lactateprepared in water (Quantity of base required to adjust pH=0.036 g). Theosmolality of this solution was 478 mOsm/kg. The final volume of thesolution was 1.7 ml. Theoretical concentration of gemifloxacin in thisformulation would be 19.95 mg/ml.

Gemofloxacin Formulation with Sodium Acetate

50.47 mg of Gemifloxacin mesylate (equivalent to 40.50 mg Gemifloxacin)was added to 2.0 ml water. The resulting solution was cloudy. It wasfiltered through a 0.45 micron filter. 1.77 ml of solution was obtainedafter filtering having a pH of 4.40. The pH of this solution wasadjusted to 5.40 with 50 uL of a 10% solution of sodium acetate preparedin water (Quantity of base required to adjust pH=0.005 g). Theosmolality of this solution was 192 mOsm/kg. The osmolality of thissolution was adjusted to 295 mOsm/kg using 28 uL of 20% solution ofsodium chloride prepared in water.

Gemofloxacin Formulation with Sodium Propionate

50.00 mg of Gemifloxacin mesylate (equivalent to 40.13 mg Gemifloxacin)was added to 1.9 ml water. The resulting solution was cloudy. It wasfiltered through a 0.45 micron filter. 1.39 ml of solution was obtainedafter filtering having a pH of 4.32. The pH of this solution wasadjusted to 5.50 with 30 uL of a 20% solution of sodium propionateprepared in water (Quantity of base required to adjust pH=0.006 g). Theosmolality of this solution was 183 mOsm/kg. The osmolality of thissolution was adjusted to 296 mOsm/kg using 25 uL of 22% solution ofsodium chloride prepared in water. This solution was remade withosmolality adjustment to 237 m0 sm/Kg

Gemifloxacin Formulation with Sodium Citrate

49.92 mg of Gemifloxacin mesylate (equivalent to 40.06 mg Gemifloxacin)was added to 1.9 ml water. The resulting solution was cloudy. It wasfiltered through a 0.45 micron filter. 1.63 ml of solution was obtainedafter filtering having a pH of 4.20. The pH of this solution wasadjusted to 5.39 with 15 uL of a 20% solution of sodium citrate preparedin water (Quantity of base required to adjust pH=0.003 g).

Example 14 Microspheres of Levofloxacin

The goal of this study was to prepare various microsphere forms oflevofloxacin that may gain taste-masking, and AUC shape-enhancingproperties through decreased solubility and/or dissolution. Thesebenefits may enhance the pharmacodynamic properties of levofloxacinfollowing pulmonary administration using either nanoparticle suspensionor dry powder inhalation. These formulations are being optimized toprolong the release of levofloxacin from decreased solubility ordissolution forms. These properties may also be incorporated into otherfluoroquinolone antibiotics including, without limitation gemifloxacin,gatifloxacin, norfloxacin, tosufloxacin, sitafloxacin sarafloxacin,prulifloxacin, and pazufloxacin. Studies are underway to characterizemicorspheres of gemifloxacin for taste masking, AUC shape-enhancement,nanoparticle suspension and dry powder inhalation administration. Otherapproaches for dry powder administration currently being investigatedinclude spray-dry and in situ micronization techniques.

Preformulation for Levofloxacin

Preformulation Studes

Preformulation studies were performed to determine the solubility oflevofloxacin and polymers in various solvents which are expected to beused during processing.

Preparation of Microspheres

A spray drying technique is being used to formulate polymermicroparticles loaded with levofloxacin. Formulation of microsphereswill typically involve dissolving the drug and polymer in a suitablesolvent. The solution is being spray dried using a spray dryer toevaporate the solvent, thereby entrapping the drug in a polymer matrix.Optimization of formulation parameters (drug: polymer ratio, polymersolution concentration and production parameters) is being performed toachieve a desired micro particle size, optimum drug loading and in vitrodrug release.

Characterization of Microspheres

The microparticles will be characterized for their morphology using SEMwhile microscopy or a suitable technique (Laser diffraction) will beused for estimating their size.

Drug loading will be determined by extracting the drug from themicrospheres in a suitable solvent and analyzing the extract by UV/HPLC.

Drug release from the microspheres will be carried out using a USPdissolution apparatus.

Example 15 Inhalation Toxicology in Rats

In a 4 day non-GLP ascending dose study of aerosolized levofloxacin inmale and female Sprague-Dawley rats, a 25 mg/ml solution of levofloxacinwas administered for one hour on day one and a 50 mg/ml solution oflevofloxacin was administered for two hours per day on days 2 thru 4. Noclinical signs of toxicity were observed during the treatment period.Necropsy 24 hours after administration of the last dose did not show anyfindings.

In a GLP study of aerosolized levofloxacin in male and femaleSprague-Dawley rats, aerosolized levofloxacin was administered dailywith an average dose of 6.92 mg/kg/day to the males and 10.04 mg/kg/dayfor the females over 4 days using a nose-only aerosol delivery device.Total exposures were 29 and 42 mg/kg for males and females, respectivelyover the study period. Each dose was delivered over 2 hours daily. Thedose for this study was chosen based on the maximum solubility oflevofloxacin that could be administered in the device over 2 hours. Noclinical signs of toxicity were observed, and all animals survivedduring the 4 day treatment period. Necropsy of animals afteradministration of the last dose did not show any findings.

In a 28-day GLP study in Sprague-Dawley rats, animals were randomized to3 dose levels of aerosolized levofloxacin or saline. Additional recoverygroups using the vehicle control and the highest dose were also treatedand observed for a 14 day recovery period following the last dose.Average aerosolized levofloxacin doses were 1.49, 3.63, and 7.29mg/kg/day for male rats, and 2.20, 5.35, and 11.01 mg/kg/day in femalerats. The total exposures over the 28-day treatment period rangedbetween 41.7 and 204.1 mg/kg for males and 61.6 and 308.3 mg/kg forfemales. Each dose was delivered over 2 hours daily. No dose relatedclinical signs of toxicity were observed, and all animals survivedduring the 28 day treatment period. Necropsy of animals afteradministration of the last dose showed a dose related squamous cellhyperplasia of the larynx which declined in severity during a 14 dayrecovery period.

What is claimed is:
 1. A method of treating a bacterial lung infectionin a subject with sinusitis in need thereof comprising administering tothe subject with sinusitis a therapeutically effective amount of anaerosol of a solution comprising about 100 mM to about 625 mM oflevofloxacin or ofloxacin and about 25 mM to about 400 mM of a divalentor trivalent cation, to treat the bacterial lung infection.
 2. Themethod of claim 1, wherein the solution has a pH from about 5.5 to about6.5, and an osmolality from about 350 mOsmol/kg to about 750 mOsmol/kg.3. The method of claim 1, wherein the divalent or trivalent cation iscalcium, iron, or zinc.
 4. method of claim 1, wherein the divalent ortrivalent cation is magnesium.
 5. The method of claim 4, wherein themagnesium is magnesium chloride.
 6. The method of claim 5, wherein thesolution comprises about 100 mM to about 625 mM levofloxacin and about25 mM to about 400 mM magnesium chloride; and wherein the solution has apH from about 5.5 to about 6.5, and an osmolality from about 350mOsmol/kg to about 750 mOsmol/kg.
 7. The method of claim 1, wherein thesolution comprises about 100 mM to about 625 mM levofloxacin and about25 mM to about 400 mM of a divalent or trivalent cation selected fromthe group consisting of magnesium, calcium, iron, and zinc.
 8. Themethod of claim 1, wherein the lung infection is caused by a gramnegative bacteria, a gram negative anaerobic bacteria, a gram positivebacteria, a gram positive anaerobic bacteria, or a combination of two ormore thereof.
 9. The method of claim 1, wherein the lung infection iscaused by one or more bacteria selected from the group consisting ofPseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonasacidovorans, Pseudomonas alcaligenes, Pseudomonas putida,Stenotrophomonas maltophilia, Aeromonas hydrophilia, Escherichia coli,Citrobacter freundii, Salmonella typhimurium, Salmonella typhi,Salmonella paratyphi, Salmonella enteritidis, Shigella dysenteriae,Shigella flexneri, Shigella sonnei, Enterobacter cloacae, Enterobacteraerogenes, Klebsiella pneumoniae, Klebsiella oxytoca, Serratiamarcescens, Morganella morganii, Proteus mirabilis, Proteus vulgaris,Providencia alcalifaciens, Providencia rettgeri, Providencia stuartii,Acinetobacter calcoaceticus, Acinetobacter haemolyticus, Yersiniaenterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Yersiniaintermedia, Bordetella pertussis, Bordetella parapertussis, Bordetellabronchiseptica, Pasteurella multocida, Pasteurella haemolytica,Helicobacter pylori, Campylobacter fetus, Campylobacter jejune,Campylobacter coli, Borrelia burgdorferi, Vibrio cholera, Vibrioparahaemolyticus, Legionella pneumophila, Listeria monocytogenes,Neisseria gonorrhoeae, Neisseria meningitidis, Burkholderia cepacia,Francisella tularensis, Kingella, and Moraxella.
 10. The method of claim1, wherein the lung infection is caused by one or more bacteria selectedfrom the group consisting of Pseudomonas aeruginosa, Moraxella,Haemophilus influenzae, Haemophilus parainfluenzae, Haemophilushaemolyticus, Haemophilus parahaemolyticus, and Haemophilus ducreyi. 11.The method of claim 1, wherein the lung infection is caused by one ormore bacteria selected from the group consisting of Corynebacteriumdiphtherias, Corynebacterium ulcerans, Streptococcus pneumoniae,Streptococcus agalactiae, Streptococcus pyogenes, Streptococcus milleri;Streptococcus (Group G); Streptococcus (Group C/F); Enterococcusfaecalis, Enterococcus faecium, Staphylococcus aureus, Staphylococcusepidermidis, Staphylococcus saprophyticus, Staphylococcus intermedius,Staphylococcus hyicus subsp. hyicus, Staphylococcus haemolyticus,Staphylococcus hominis, and Staphylococcus saccharolyticus.
 12. A methodof treating sinusitis in a subject in need thereof comprisingadministering to the subject a therapeutically effective amount of anaerosol of a solution comprising about 100 mM to about 625 mMlevofloxacin or ofloxacin and about 25 mM to about 400 mM of a divalentor trivalent cation to treat the sinusitis.
 13. The method of claim 12,wherein the solution has a pH from about 5.5 to about 6.5, and anosmolality from about 350 mOsmol/kg to about 750 mOsmol/kg.
 14. Themethod of claim 12, wherein the divalent or trivalent cation is calcium,iron, or zinc.
 15. The method of claim 12, wherein the divalent ortrivalent cation is magnesium.
 16. The method of claim 15, wherein themagnesium is magnesium chloride.
 17. The method of claim 16, wherein thesolution comprises about 100 mM to about 625 mM levofloxacin and about25 mM to about 400 mM magnesium chloride; and wherein the solution has apH from about 5.5 to about 6.5, and an osmolality from about 350mOsmol/kg to about 750 mOsmol/kg.
 18. The method of claim 12, whereinthe solution comprises about 100 mM to about 625 mM levofloxacin andabout 25 mM to about 400 mM of a divalent or trivalent cation selectedfrom the group consisting of magnesium, calcium, iron, and zinc.
 19. Themethod of claim 12, wherein the subject has a bacterial lung infectioncaused by a gram negative bacteria, a gram negative anaerobic bacteria,a gram positive bacteria, a gram positive anaerobic bacteria, or acombination of two or more thereof.
 20. The method of claim 12, whereinthe subject has a bacterial lung infection caused by one or morebacteria selected from the group consisting of Pseudomonas aeruginosa,Pseudomonas fluorescens, Pseudomonas acidovorans, Pseudomonasalcaligenes, Pseudomonas putida, Stenotrophomonas maltophilia, Aeromonashydrophilia, Escherichia coli, Citrobacter freundii, Salmonellatyphimurium, Salmonella typhi, Salmonella paratyphi, Salmonellaenteritidis, Shigella dysenteriae, Shigella flexneri, Shigella sonnei,Enterobacter cloacae, Enterobacter aerogenes, Klebsiella pneumoniae,Klebsiella oxytoca, Serratia marcescens, Morganella morganii, Proteusmirabilis, Proteus vulgaris, Providencia alcalifaciens, Providenciarettgeri, Providencia stuartii, Acinetobacter calcoaceticus,Acinetobacter haemolyticus, Yersinia enterocolitica, Yersinia pestis,Yersinia pseudotuberculosis, Yersinia intermedia, Bordetella pertussis,Bordetella parapertussis, Bordetella bronchiseptica, Pasteurellamultocida, Pasteurella haemolytica, Helicobacter pylori, Campylobacterfetus, Campylobacter jejune, Campylobacter coli, Borrelia burgdorferi,Vibrio cholera, Vibrio parahaemolyticus, Legionella pneumophila,Listeria monocytogenes, Neisseria gonorrhoeae, Neisseria meningitidis,Burkholderia cepacia, Francisella tularensis, Kingella, and Moraxella.21. The method of claim 12, wherein the subject has a bacterial lunginfection caused by one or more bacteria selected from the groupconsisting of Pseudomonas aeruginosa, Moraxella, Haemophilus influenzae,Haemophilus parainfluenzae, Haemophilus haemolyticus, Haemophilusparahaemolyticus, and Haemophilus ducreyi.
 22. The method of claim 12,wherein the subject has a bacterial lung infection caused by one or morebacteria selected from the group consisting of Corynebacteriumdiphtherias, Corynebacterium ulcerans, Streptococcus pneumoniae,Streptococcus agalactiae, Streptococcus pyogenes, Streptococcus milleri;Streptococcus (Group G); Streptococcus (Group C/F); Enterococcusfaecalis, Enterococcus faecium, Staphylococcus aureus, Staphylococcusepidermidis, Staphylococcus saprophyticus, Staphylococcus intermedius,Staphylococcus hyicus subsp. hyicus, Staphylococcus haemolyticus,Staphylococcus hominis, and Staphylococcus saccharolyticus.